Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex. In bacteria, where the fatty acids are used primarily for incorporation into components of cell membranes, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction. The initiation of the elongation step, which extends the length of the growing acyl chain by two carbons, requires the transfer of the malonyl moiety from malonyl-CoA onto the acyl carrier protein. We report here the crystal structure (refined at 1.5-A resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from Escherichia coli. The protein has an alpha/beta type architecture, but its fold is unique. The active site inferred from the location of the catalytic Ser-92 contains a typical nucleophilic elbow as observed in alpha/beta hydrolases. Serine 92 is hydrogen bonded to His-201 in a fashion similar to various serine hydrolases. However, instead of a carboxyl acid typically found in catalytic triads, the main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an interaction with His-201. Two other residues, Arg-117 and Glu-11, are also located in the active site, although their function is not clear.

Summary. Lipid production of the oleaginous yeast Apiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h -1. At C/N-ratios higher than 25-30 lipid content gradually increased to nearly 50% at C / N = 70 and the maximal obtainable dilution rate decreased to 0.02 h -1 at C / N = 7 0 . From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30-35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates of A. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes

SummaryWe demonstrate that fluorescent proteins can be used as visual selection markers for the

A mathematical model was constructed to describe the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production by oleaginous yeasts. To test this model and to determine some relevant model parameters, the oleaginous yeast Apiotrichum curvatum ATCC 20509 was grown in continuous cultures at various C/N-ratios and dilution rates. It appeared that when nitrogen is limiting for the formation of biomass, the remaining glucose can be converted to storage carbohydrate and storage lipid. No clear dependence of carbohydrate yield on the C/N-ratio could be demonstrated, but lipid yield increased gradually with increasing C/N-ratios. The maximal dilution rate for lipid producing yeast cells appeared to be optimal at relatively low C/N-ratios. It can be concluded that the experimental results fitted well with the mathematical model. By using this model, lipid yield and lipid production rate can be calculated at any C/N-ratio of the growth medium and optimum operation conditions can be predicted for the production of microbial lipids.

We report the cloning and nucleotide sequence of the gene encoding malonyl cocnzymc A-acyl carrier protein transacylasc of Esciwrichiu co/i. Malonyl transacylasc has been overexpressed 1.55.fold compared to a wild-type strain, Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of ris-vacccnate arc incorporated into the mcmbranc phospholipids. Mulonyl transacylase; Malonyl-CoA: Acyl transferase; Fatty acid biosynthesis 1. 1NTRODUCTION Malonyl coenzyme A-acyl carrier protein trans-acylase (malonyl transacylase) catalyzes a key reaction of fatty acid synthesis in bacteria and plants, the conversion of malonyl-CoA to malonyl-acyl carrier protein (ACP). Malonyl-ACP then acts as the two carbon donor in the elongation steps of fatty acid synthesis. A similar reaction occurs in fatty acid synthesis in fungi and mammals except that both the malonyl transacylase and ACP moieties are domains of the polyfunctional fatty acid synthases of these organisms. E. co/i malonyl transacylase has been purified [1,2], and aspects of the enzymatic mechanism studied [3]. Mutants defective in the enzyme have also been isolated [4], In this paper, we report the cloning, DNA sequence, and overexpression of malonyl transacylase. We also report the effects of overexpression on the fatty acid composition of the membrane phospholipids of E. co/i, 2. MATERIALS AND METHODS The bacterial strains used were all derivatives of E. ru/i K-l?. Strain L48 (also called LA2-89) [4], carries a temperature-sensitive lesion in the&&I gene resulting in a malonyl transacylaseol'abnormal thermo-lability. recA derivatives of this strain wrrc made tither by PI transduction or bacterial conjugation using a T~rrfO element closely linked to rccAf. Standard recombinant DNA methods were used as were standard plasmids and host strains. DNA sequencing was done using the Sequenasc kits from United States Bibchemicals with both single-stranded and double-stranded templates. The sequence reported was obtained by complete sequencing of both DNA strands and was independently obtained in both laboratories. Protein cxpres-C~r-esfi~rrlen~ &ifzrz. sion studies were done using either the T7 polymerase system (for the truncated protein) [S], or the f~ promoter of plasmid pCKRlOl [6]. 3. RESULTS 3.1. Sequejrce of fabD The fobD gene was isolated from two different sources; lambda miniset phase 235 of the Kohara library [7] and from a minibank of E. co/i DNA fragments in plasmid pACYCl84 [El. In both cases, the gene was isolated by complementation (or recombinational repair) of theJrbl> strain LA2-89 which encodes a temperature sensitive enzyme. The gene carried by a235 was further localized by subcloning into high copy number plasmids. The minimal clone conferring growth at 42°C was sequenced and found to encode a truncated version of FabD lacking the last 21 amino acids. N-terminal sequencing of the truncated protein gave the sequence TQFAFVFPGQ, and thus the initiation me-thionine is the most upstream of the three in-fr...