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Four novel proteasome inhibitors, TMC-95A-D (1-4) have been isolated from the fermentation broth of Apiospora montagnei Sacc. TC 1093, isolated from a soil sample. All of the molecular formulas of 1-4 were established as C(33)H(38)N(6)O(10) by high-resolution FAB-MS. Their planar structures were determined on the basis of extensive analyses of 1D and 2D NMR, and degradation studies. Compounds 1-4 have the same planar structures to each other, and are unique highly modified cyclic peptides containing L-tyrosine, L-aspargine, highly oxidized L-tryptophan, (Z)-1-propenylamine, and 3-methyl-2-oxopentanoic acid units. The absolute configuration at C-11 and C-36 of 1-4 was determined based on chiral TLC and HPLC analyses of their chemical degradation products. The ROESY analysis along with (1)H-(1)H coupling constants clarified the absolute stereochemistry at C-6, -7, -8, and -14 of the cyclic moieties. These studies revealed the relationships of 1-4 to be diastereomers at C-7 and C-36.

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The proBA operon, coding for y-glutamyl b a s e (GK) and y-glutamyl phosphate reductase (GPR), was cloned from the chromosome of the wild-type strain of Serratia marcescens Sr41. From our sequence data the proB (1101 bp) and proA (1472 bp) genes were shown to code for two proteins of M, 39169 and 44640, respectively. Analysis of expression of the ZacZstructural gene fused with theproBA promoter showed that theproBA operon is not subject to proline-mediated feedback repression. Amplification of the proBA operon enabled us to determine GK activity, which was inhibited in the presence of a low concentration of L-proline. Comparison of the amino acid sequences of the S. marcescens GK and GPR proteins with those of the GI( and GPR proteins from E. coZi revealed extensive similarities.

The polyether-macrolide antibiotic, boromycin, was isolated as a potent anti-human immunodeficiency virus (HIV) antibiotic from a fermentation broth of Streptomyces sp. A-3376. Boromycin was found to strongly inhibit the replication of the clinically isolated HIV-1 strain as well as the cultured strain in in vitro laboratory experiments. The mechanism for the anti-HIV activity of boromycin is suggested to involve blocking the later stage of HIV infection, and probably the maturity step for replication of the HIV molecule.

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The nucleotide sequence of the proBA operon from a proline-hyperproducing mutant of Serratia marcescens was determined. Two base substitutions were found: one in theproBstructura1 gene, coding for y-glutamyl kinase (GK), and a second one in the promoter region of the operon. The former base substitution led to a change of the predicted amino acid at position 117 from an alanine to a valine in GK. This mutation rendered GK 700-fold less sensitive to proline-mediated feedback inhibition than the wild-type enzyme. The other base substitution, a transversion from a G-C to an A-T, was located in the spacer region between the ' -35' and ' -10' sequences of the promoter, and it increased the transcriptional activity of this operon fourfold. Both these two base substitutions, which were acquired at the step of selecting mutants resistant to a toxic proline analogue, 3,4-dehydroproline, confer upon cells a high proline productivity and an increased osmotolerance.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonasfluorescens B52 and P.fluorescens SIK Wi, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.Triacylglycerol acylhydrolase (EC 3.1.1.3) catalyzes the hydrolysis of triacylglycerol to glycerol and fatty acid and is generally called lipase. This enzyme is important in the food industry, and there is also current interest in the application of lipases to the production of chiral compounds. Extracellular lipases are produced by a variety of microorganisms: fungi, yeasts, and bacteria, including actinomycetes (60, 64). The lipase genes have been cloned from fungi (4, 21, 57, 58, 66), a yeast (32), Pseudomonas species (1, 6, 27, 29, 31, 34, 61a, 65), and staphylococci (19, 38). Serratia marcescens, an enteric bacterium, produces several extracellular enzymes which participate in the degradation of high-molecular-weight compounds, e.g., serine protease (67), chitinase (30), phospholipase (17), and nuclease (2, 7). Their genes have been cloned from S. marcescens, and the deduced amino acid sequences indicated that these proteins are secreted through a mechanism containing the secAY gene products with the aid of typical N-terminal signal peptides as described previously (51). Interestingly, the S. marcescens metalloprotease has been recently reported to be excreted through a mechanism similar to that of Escherichia coli ot-hemolysin having no N-terminal signal peptide (40).S. marcescens has been long known to produce extracellular lipase (64), but there is little knowledge of its enzyme. Very recently, we showed it to be stable in some organic solvents and to be applicable to the synthesis of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester by asymmetric hydrolysis of racemic trans-3-(4-methoxyphenyl)glycidic acid methyl ester, a key intermediate in the synthesis of diltiazem hydrochloride that is useful as a coronary vasodilator (44). Our final goal is the construction of a lipase-hyperproducing strain of S. marcescens. This paper deals with the molecular cloning of the S. MATERIALS AND METHODSStrains and plasmids. E. coli K-12 DH5 (53) was used as a host for the construction of plasmids. The wild-typ...