Klebsiella aerogenes ATCC 9621 was able to utilize phosphonates (P_n), including aminoethylphosphonate, ethylphosphonate, methylphosphonate (MP_n), and phosphonoacetate, and inorganic phosphite (P_t) as sole sources of phosphorus (P). The products of the phn gene cluster were absolutely required for P_n breakdown and P_t oxidation to inorganic phosphate (P_i) in this organism. To determine if K. aerogenes ATCC 9621 could be engineered to enhance the utilization of P_n and P_t, a multicopy plasmid, pBI05, which carried the entire phn gene cluster, was introduced into this strain. Despite the increased dosage of the phn genes, K. aerogenes ATCC 9621(pBI05) could utilize only up to 1.1-fold more P_n and P_t than did the control strain with the parent vector alone. These results suggested that P_i, which was generated from P_n and P_t, might limit further utilization of these P compounds. Consequently, to convert the resulting P_i to polyphosphate (polyP), the plasmid pKP28, which carried the K. aerogenes ppk gene (which encodes polyP kinase), was introduced into K. aerogenes ATCC 9621(pBI05). Overexpression of the ppk gene in K. aerogenes ATCC 9621(pBI05, pKP28) resulted in a 2.5-fold increase in P_t utilization over that of the control strain. This recombinant strain also accumulated approximately sixfold more P than did the control strain when the cells were grown with MP_n as a sole source of P.