SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.

The global economy heads for a severe energy crisis: whereas the energy demand is going to rise, easily accessible sources of crude oil are expected to be depleted in only 10-20 years. Since a serious decline of oil supply and an associated collapse of the economy might be reality very soon, alternative energies and also biofuels that replace fossil fuels must be established. In addition, these alternatives should not further impair the environment and climate. About 90% of the biofuel market is currently captured by bioethanol and biodiesel. Biodiesel is composed of fatty acid alkyl esters (FAAE) and can be synthesized by chemical, enzymatic, or in vivo catalysis mainly from renewable resources. Biodiesel is already established as it is compatible with the existing fuel infrastructure, non-toxic, and has superior combustion characteristics than fossil diesel; and in 2008, the global production was 12.2 million tons. The biotechnological production of FAAE from low cost and abundant feedstocks like biomass will enable an appreciable substitution of petroleum diesel. To overcome high costs for immobilized enzymes, the in vivo synthesis of FAAE using bacteria represents a promising approach. This article points to the potential of different FAAE as alternative biofuels, e.g., by comparing their fuel properties. In addition to conventional production processes, this review presents natural and genetically engineered biological systems capable of in vivo FAAE synthesis.

Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full “overcapacity” of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be “lost.”

No abstract

No abstract

For the biotechnical production of biofuels, oleo‐, or fine chemicals bacterial wax ester synthase/acyl‐Coenzyme A:diacylglycerol acyltransferases (WS/DGAT) are discussed as interesting candidates for in vivo esterification reactions. In this study, the suitability of selected acyltransferases for the conversion of non‐physiological substrates like short‐chain‐length alcohols and short‐chain length or even branched acyl‐CoAs has been investigated. In vitro analyzes of purified AtfA and AtfA(G355I) from Acinetobacter baylyi, Ma1(A360I) from Marinobacter aquaeolei, WS2 from Marinobacter hydrocarbonoclasticus, and AtfA1 from Alcanivorax borkumensis were conducted to evaluate the specific activities of these enzymes toward n‐hexadecanol (C16), n‐dodecanol (C12), ethanol (C2), and methanol (C1), palmitoyl‐CoA (C16), butyryl‐CoA (C4) as well as toward branched 3‐hydroxybutyryl‐CoA and 2‐hydroxyisobutyryl‐CoA. Athough long‐ and medium‐chain‐length substrates were preferred by all five enzymes, WS2 and AtfA showed the highest relative activities with ethanol or methanol when compared to n‐hexadecanol, whereas residual activities toward short or branched acyl‐CoAs could only be measured with AtfA and AtfA1. Practical applications: Bacterial WS/DGATs can be used for in vivo and in vitro approaches to synthesize custom‐made lipids, such as triglycerides or wax esters. However, due to their broad substrate ranges these enzymes are also promising candidates for the synthesis of other, industrially valuable oleo‐ and fine chemicals. Short‐chain‐length esters are important intermediates and building blocks for many production processes and, at present, there is a great demand for enzymes which are able to catalyze their synthesis. The physiological substrates of bacterial WS/DGAT enzymes are medium‐ to long‐chain length acyl‐CoAs and fatty alcohols to synthesize medium‐ to long‐chain length wax esters. In this study, we investigated five different bacterial WS/DGATs for their ability to synthesize short‐chain length esters, which represent biotechnologically interesting compounds. The enzymes AtfA, AtfA(G355I), Ma1(A360I), WS2, and AtfA1 were selected, purified, and characterized in vitro. In addition to the reference substrates, hexadecanol and palmitoyl‐CoA, their activity with dodecanol, ethanol, or methanol, on the one hand, and lauryl‐CoA, butyryl‐CoA, 3‐hydroxybutyryl‐CoA, or 2‐hydroxyisobutyryl‐CoA, on the other hand, were studied and compared.

Acinetobacter baylyi synthesizes significant amounts of wax esters (WE) and triacylglycerols (TAG) catalyzed by wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT or AtfA), representing the key enzyme for bacterial lipid accumulation. However, the structure and exact biochemical mechanism of AtfA could not be elucidated, yet. Therefore, a combination of random mutagenesis, screening and sequencing of atfA gene variants was conducted to gain further insights into the relationship between sequence and function of the enzyme. Several mutations could be detected which seriously diminished lipid accumulation in A. baylyi as well as AtfA activity in recombinant E. coli strains, such as Glu15Lys, Trp67Gly, Ala126Asp, Ser374Pro, or Gly378Ser/Asp. The affected residues are more or less conserved among a wide range of AtfA homologs. Especially the highly conserved pattern SNVPGP seems to be crucial, as mutations inside this pattern drastically impair enzyme activity. Furthermore, it became obvious that the C‐terminal part of AtfA is indispensable for activity, although the catalytic core is located in the N‐terminal half of the enzyme. In silico studies suggest that the C‐terminus might form a coiled‐coil fold which could putatively represent the dimerization domain. Practical applications: WE, composed of a long‐chain acyl moiety and a fatty alcohol residue, are valuable ingredients of many commercial products like cosmetics, medical products or lubricants. However, natural sources for high‐quality WE are currently mainly restricted to the expensive oil of the jojoba plant or to carnauba wax. As A. baylyi naturally accumulates WE with a similar composition to jojoba‐oil, this organisms and the responsible acyltransferase AtfA, are of great interest regarding a sustainable biotechnological WE production. However, a lack of structural knowledge about this enzyme family currently constricts promising enzyme optimization approaches. The insights gained by random mutagenesis of AtfA can build a basis for further site‐directed mutagenesis approaches in order to optimize its activity, stability, and/or substrate range.

Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli. The purified enzyme Atf G25 showed acyltransferase activity with C 12 -or C 16 -acyl-CoA, C 12 to C 18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of Atf G25 on lipid accumulation, the respective gene, atf G25 , was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that Atf G25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCEA novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids. Even extreme environments, such as arid, desert-like regions, are populated by prokaryotes. These mostly Gram-positive bacteria are subjected to extreme fluctuations of temperature and water supply. In addition to desiccation and the resulting cellular damage, the bacteria frequently encounter nutrient limitation. To withstand these harsh environmental conditions, the majority of bacteria have evolved various strategies and are able to store lipophilic compounds such as polyhydroxyalkanoates (PHA), triacylglycerols (TAG), or wax esters (WE) (1-3). TAG are synthesized as primary storage compounds by many actinomycetes-which represent the pre...