Front Matter

DNA sequence identity within the signal, propeptide, and mature lipase regions were

47 %, 53 % and 59 %, respectively. Maximum alignment of the amino acid se-

quences of the Rd and Rm lipases was obtained by the introduction of only one

gap, a single amino acid long, in each sequence. Upon such alignment, homologies

are substantial. Considering both identical and functionally equivalent residues, the

predicted sequences are 29 % and 68 % homologous in the pre/pro and mature do-

mains, respectively. The predicted mature lipases from both organisms contain 269

amino acids. In the halves of the molecules containing the active site pentapeptide,

sequence conservation is substantially higher, with 76 % of the amino acids being

identical or functionally equivalent.

Consistent with the proposal that manyRhizopusisolates are actually the same

organism (Schipper, 1984), nearly complete homologies, and some identities,

were observed between the amino acid sequences of the Rd lipase and those pro-

duced by other members of the genusRhizopus(Kujimiya et al., 1992; Uyttenbroeck

et al., 1993; Beer et al., 1996). As further sequence and structural information be-

came available, the existence of a family of related enzymes including not only

RhizopusandRhizomucorlipases but also those produced byHumicola lanuginosa

andPenicillium camembertiibecame apparent (Derewenda et al., 1994a,b).

4.6 Subcloning and regulated overexpression

of the lipase gene

The initial Rd lipase gene clone, though expressed by itsE. colihost, did not produce

significant amounts of lipase. Furthermore, due to processing inefficiencies on the

part of the host, lipase molecules that did accumulate consisted predominantly of

larger, immature forms. To overcome these deficiencies and to generate recombi-

nant lipase comparable to that produced by the authentic fungal host, the isolated

cDNA was further engineered (Joerger and Haas, 1993). Site-directed mutagenesis

was employed to introduce a unique restriction endonuclease recognition site and a

translation initiation site just upstream of the sequence for the mature lipase. Thus, a

gene encoding the direct production of the mature lipase, containing neither signal

peptide nor propeptide, was produced. Similarly, a cDNA that encoded the prolipase,

without an attendant export signal peptide, was engineered. This was a construct of

considerable interest since it was unclear whether the prolipase was enzymatically

active, and whether it exhibited biochemical properties comparable to those of the

mature lipase.

During work with the cloned Rd lipase cDNA it became apparent that expression

of the gene had a negative effect on bacterial hosts. In some cases, plasmids contain-

ing the lipase gene displayed unexpectedly low transformation frequencies, exhib-

ited elevated instabilities, and reduced the viabilities of their hosts (W. Baker and R.

Joerger, unpublished observations). Cell lines containing the gene sometimes had

visually distinct morphologies. That a lipase could be damaging to its host if pro-

duced and held within the cell was not unexpected, given that these enzymes are

known to be active on phospholipids (Laboureur and Labrousse, 1964; Slotboom

et al., 1970), the major lipid component of the bacterial inner membrane. Thus,

78 4 Cloning, Mutagenesis, and Biochemical Properties