Evaluation of strains derived from Escherichia coli W as hosts for the expression of penicillin G-acylase-encoding gene cloned on the recombinant plasmid pKA18.

The potential for production of penicillin G-acylase (PGA), encoded by the chromosomal gene pgai, of four strains belonging to a genealogical line derived from the strain Escherichia coli W, was evaluated in a medium with and without the inducer phenylacetic acid (PA). These strains were used as hosts of the recombinant plasmid pKA18, in which the structural gene pgac isolated from the strain RE3, the best host strain of a line giving the highest production, was cloned. The presence of the inducer reduced the copy number of the plasmid in all recombinant strains. Only in recombinant strain RE3 (pKA18) the reduction of the gene dosage of pgac resulted also in the reduction of the amount of PGA synthesized by the cells. The reduced activity of the cells did not result from a segregation of plasmid-free clones. Also the growth rate was decreased by 20 and 40% in the host and recombinant strains, respectively. The host strain RE3 showing the highest production of PGA was also the best host of the recombinant plasmid in terms of the segregational stability and copy number (198 copies per chromosome). The recombinant strain RE3 (pKA18) also provided the highest production of PGA.

[Penicillin G acylase--synthesis, regulation, production]. / Penicilin G acylasa--syntéza, regulace, produkce.

Penicillin G acylase (PGA) is one of very important industrial enzymes used in the production of polysynthetic beta-lactam antibiotics. 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 chromozome and synthesized as the pre-pro-PGA (pp PGA) precursor, which is transported, 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 alpha and beta. 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 transform suitable host strains, some of which are now used in industry as highly productive microorganisms.

Indigenous plasmids in a production line of strains for penicillin G acylase derived from Escherichia coli W.

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase (PGA) derived from the strain Escherichia coli W ATCC 9637. Their size and copy number (CN) in E. coli W were determined (kb; CN): pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). Strain E. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.

Plasmid burden in chemostat culture of Escherichia coli: Its effect on the selection for overproducers of host enzymes.

The effect of plasmid-mediated metabolic burden of on the expression of the host genes and its consequences on the plasmid maintenance were studied in carbon-limited chemostat culture of Escherichia coli 1EA(pBR322) subject to selection for strains overproducing chromosomally coded ribitol dehydrogenase. The chemostat population became rapidly heterogeneous and the competition among evolved strains was found to be crucial for the kinetics of the plasmid loss from the culture. The selective disadvantages in growth rate associated with plasmid carriage in the parent-like and ribitol dehydrogenase-overproducing strains was estimated.

Modified method of application of ethyl methanesulfonate for induction of Escherichia coli mutants.

An efficient modification of ethyl methanesulfonate mutagenic action is based on mutagenization of small volumes of cell suspensions in micro-sample tubes. This provides for a rapid and safe handling of solutions of cancerogenic mutagens. A 3-4-h exposition to 30-40 mmol/L of mutagen appeared optimal, inducing more than 20% auxotrophs or, after a simultaneous application of the penicillin method, 60% of auxotrophs. Moreover, the modification proved its value in repeated applications of ethyl methanesulfonate, resulting in an accumulation of various mutation types. Consecutive mutations were accompanied by an increase in sensitivity. Based on the distribution of nucleoids in the mutagenized population, the penicillin method was modified to allow detection of mutants segregated from cells with several nucleoids.