We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

A clinical isolate ofPseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a 1-lactamase. This f-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pl of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pl of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km)* PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type 13-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum 13-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.More than 50 biochemically distinct P-lactamases responsible for resistance to ,-lactams have been reported in gram-negative bacteria. The resistance of broad-spectrum cephalosporins to these ,B-lactamases was a widely accepted concept in the beginning of the 1980s. However, overproduction of chromosomally mediated cephalosporinases has been described as responsible for failure of treatment of gram-negative bacterial infections with extended-spectrum cephalosporins (39). Since 1983, plasmid-mediated extended-spectrum 3-lactamases have been reported, primarily in Kiebsiella pneumoniae and then in numerous Enterobacteraceae species (16, 34). These enzymes hydrolyze extended-spectrum cephalosporins and aztreonam to various extents but usually neither cephamycins (cefoxitin and moxalactam) nor carbapenems (imipenem and meropenem). A common feature of these enzymes is inhibition of their activity by clavulanic acid. These enzymes are Ambler class A 1-lactamases, members of the TEM or SHV series that differ by a few point mutations in their structural genes (16,34 resistance to extended-spectrum cephalosporins. In this species, TEM-1 and TEM-2 1-lactamases confer additional resistance to ureidopenicillins (26). The OXA-type (oxacillin-hydrolyzing) enzymes possess high-level hydrolytic activity against cloxacillin, oxacillin, and methicillin (9, 10). Their activities are inhibited by clavulanic acid but to a lesser extent than TEM-or SHV-der...