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The ability of Escherichia coli MV1184 to accumulate inorganic phosphate (Pi) was enhanced by manipulating the genes involved in the transport and metabolism of Pi. The high-level Pi accumulation was achieved by modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate-inducible transport system (pstS, pstC, pstA, and pstB). Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. Recombinant strains, which contained either pBC29 (carrying ppk) or pEP02.2 (pst operon), removed approximately twoand threefold, respectively, more P; from minimal medium than did the control strain. The highest rates of Pi removal were obtained by strain MV1184 containing pEP03 (ppk and ackA). However, unlike the control strain, MV1184(pEP03) released Pi to the medium after growth had stopped. Drastic changes in growth and Pi uptake were observed when pBC29 (ppk) and pEP02.2 (pst operon) were introduced simultaneously into MV1184. Even though growth of this recombinant was severely limited in minimal medium, the recombinant could remove approximately threefold more Pi than the control strain. Consequently, the phosphorus content of this recombinant reached a maximum of approximately 16% on a dry weight basis (49%o as phosphate). * Corresponding author. also has been found downstream of the ppk gene, constituting a polyP operon (2a). Under ordinary operating conditions, activated sludges are capable of removing an average of only 20 to 40% of the

Genetic improvement of bacterial ability to accumulate phosphate (Pi) was investigated using Escherichia coli as a test organism. High levels of Pi accumulation were achieved by (i) modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate inducible transport system (pstS, pstC, pstA, and pstB) and (ii) genetically inactivating ppx encoding exopolyphosphatase. Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. The best recombinant strain, which contained both pBC29 (ppk) and pEP02.2 (pst genes) accumulated approximately 10-fold more Pi than did the control strain. The phosphorus content of this recombinant reached a maximum of 16 % on the dry weight basis (49 % as phosphate). About 65 % of the cellular phosphorus was stored as polyphosphate.

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