Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis.

Biosynthesis of the polyketide-derived carbon skeleton of daunorubicin (DNR) begins with propionate rather than acetate, which is the starter unit for most other aromatic polyketides. The dpsCgene has been implicated in specifying the unique propionate-starter unit, and it encodes a protein that is very similar to the Escherichia coli beta-ketoacyl:acyl carrier protein (ACP) synthase III (FabH or KS III) enzyme of fatty acid biosynthesis. Purified DpsC was found to use propionyl-coenzyme A as substrate and to be acylated by propionate at the Ser-118 residue. DpsC exhibits KS III activity in catalyzing the condensation of propionyl-CoA and malonyl-ACP, and also functions as an acyltransferase in the transfer of propionate to an ACP. The DpsC enzyme has a high-substrate specificity, utilizing only propionyl-CoA, and not malonyl-CoA, 2-methylmalonyl-CoA or acetyl-CoA, as the starter unit of DNR biosynthesis.     

The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5

The biosynthesis of daunorubicin and its precursors proceeds via the condensation of nine C-2 units derived from malonyl-CoA onto a propionyl starter moiety. The daunorubicin polyketide biosynthesis gene cluster of Streptomyces sp. strain C5 has two unique open reading frames, dpsC and dpsD, encoding, respectively, a fatty acid ketoacyl synthase (KAS) III homologue that is lacking an active-site cysteine and a proposed acyl-CoA:acyl carrier protein acyltransferase. The two genes are positioned directly downstream of dpsA and dpsB which encode the alpha and beta components of the type II KAS, respectively. Expression of the dpsABCDEFGdauGI genes in Streptomyces lividans resulted in the formation of aklanonic acid, the first stable chromophore of the daunorubicin biosynthesis pathway. Deletion of dpsC, but not dpsD, from this gene set resulted in the formation of desmethylaklanonic acid, derived from an acetyl-CoA starter unit, and aklanonic acid, derived from propionyl-CoA, in a 60:40 ratio. Thus, DpsC contributes to the selection of propionyl-CoA as the starter unit but does not alone dictate it. A dpsCD deletion mutant of Streptomyces sp. strain C5 (C5VR5) still produced daunorubicin but, more significantly, anthracycline and anthracyclinone derivatives resulting from the use of acetyl-CoA as an alternative starter moiety. Expression of dpsC, but not dpsD, in mutant C5VR5 restored the wild-type phenotype. Among the new compounds was the new biosynthesis product feudomycin D. These results suggest that in the absence of DpsC, the daunorubicin PKS complex behaves promiscuously, utilizing both acetyl-CoA (ca. 60% of the time) and propionyl-CoA (ca. 40%) as starter units. The fact that DpsC is not required for initiation with propionyl-CoA is significant, as the information must then lie in other components of the PKS complex. We propose to call DpsC the propionyl starter unit "fidelity factor." Copyright 2001 Academic Press.     

工程大肠杆菌合成游离脂肪酸的研究进展

游离脂肪酸作为一种重要的平台化合物,其衍生产品被广泛应用到能源、化学工业中。作为更加可持续、绿色的生产策略,利用工程微生物合成游离脂肪酸是以石油基和动植物为原料生产脂肪酸类产品的重要补充。大肠杆菌作为经典的模式微生物,通过对其进行代谢工程改造,脂肪酸的积累已经从痕量提高到了约9g/L,展示了其作为脂肪酸合成菌株的巨大应用潜力。随着合成生物学技术的涌现,“感应-调控器”、体外重构、β氧化逆循环、异源合成途径的整合等思路的引入极大地加快了工程大肠杆菌脂肪酸合成的进化速率,并赋予大肠杆菌合成多种脂肪酸产品的能力。对近年来通过代谢工程和合成生物学手段改造大肠杆菌合成游离脂肪酸的研究进展进行综述,对其发展前景进行展望。     

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.     

The Streptomyces peucetius dpsC Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of Streptomyces peucetius, directly downstream of the genes encoding the beta-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC and dpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.     

Metabolic engineering for the production of clinically important molecules: Omega-3 fatty acids, artemisinin, and taxol

Driven by requirements for sustainability as well as affordability and efficiency, metabolic engineering of plants and microorganisms is increasingly being pursued to produce compounds for clinical applications. This review discusses three such examples of the clinical relevance of metabolic engineering: the production of omega-3 fatty acids for the prevention of cardiovascular disease; the biosynthesis of artemisinic acid, an anti-malarial drug precursor, for the treatment of malaria; and the production of the complex natural molecule taxol, an anti-cancer agent. In terms of omega-3 fatty acids, bioengineering of fatty acid metabolism by expressing desaturases and elongases, both in soybeans and oleaginous yeast, has resulted in commercial-scale production of these beneficial molecules. Equal success has been achieved with the biosynthesis of artemisinic acid at low cost for developing countries. This is accomplished through channeling the flux of the isoprenoid pathway to the specific genes involved in artemisinin biosynthesis. Efficient coupling of the isoprenoid pathway also leads to the construction of an Escherichia coli strain that produces a high titer of taxadiene-the first committed intermediate for taxol biosynthesis. These examples of synthetic biology demonstrate the versatility of metabolic engineering to bring new solutions to our health needs.     

In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes

Abstract The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyryl-CoA and isobutyryl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyryl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA.     

Microbial production of volatile fatty acids: current status and future perspectives

Volatile fatty acids (VFAs) are used as building blocks to synthesize a wide range of commercially-important chemicals. Microbially produced VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid) can be considered as a replacement for petroleum-based VFAs due to their renewability, degradability, and sustainability. The main objective of this review is to summarize research and development of VFA production methods via microbial routes, their downstream processes, current applications, and main challenges. Various fermentation processes have been developed to produce of VFAs starting from commercially-available sugars and other raw materials such as lignocellulose, whey, and waste sludge. Only few microbes have been explored for their potential to produce VFAs, and very little genomic information data is available at the present time. There is a need to use metabolic engineering, systematic biology, evolutionary engineering, and bioinformatics to discover VFA biosynthesis routes since the pathways for isobutyric acid and isovaleric acids are still not well understood.     

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Connection of propionyl-CoA metabolism to polyketide biosynthesis in Aspergillus nidulans

Propionyl-CoA is an intermediate metabolite produced through a variety of pathways including thioesterification of propionate and catabolism of odd chain fatty acids and select amino acids. Previously, we found that disruption of the methylcitrate synthase gene, mcsA, which blocks propionyl-CoA utilization, as well as growth on propionate impaired production of several polyketides-molecules typically derived from acetyl-CoA and malonyl-CoA-including sterigmatocystin (ST), a potent carcinogen, and the conidiospore pigment. Here we describe three lines of evidence that demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. First, inactivation of a putative propionyl-CoA synthase, PcsA, which converts propionate to propionyl-CoA, restored polyketide production and reduced cellular propionyl-CoA content in a DeltamcsA background. Second, inactivation of the acetyl-CoA synthase, FacA, which is also involved in propionate utilization, restored polyketide production in the DeltamcsA background. Third, fungal growth on several compounds (e.g., heptadecanoic acid, isoleucine, and methionine) whose catabolism includes the formation of propionyl-CoA, were found to inhibit ST and conidiospore pigment production. These results demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis.     

Catabolism of leucine to branched-chain fatty acids in Staphylococcus xylosus

AIMS: Staphylococcus xylosus is an important starter culture in the production of flavours from the branched-chain amino acids leucine, valine and isoleucine in fermented meat products. The sensorially most important flavour compounds are the branched-chain aldehydes and acids derived from the corresponding amino acids and this paper intends to perspectivate these flavour compounds in the context of leucine metabolism. METHODS AND RESULTS: GC and GC/MS analysis combined with stable isotope labelling was used to study leucine catabolism. This amino acid together with valine and isoleucine was used as precursors for the production of branched-chain fatty acids for cell membrane biosynthesis during growth. A 83.3% of the cellular fatty acids were branched. The dominating fatty acid was anteiso-C(15:0) that constituted 55% of the fatty acids. A pyridoxal 5'-phosphate and alpha-ketoacid dependent reaction catalysed the deamination of leucine, valine and isoleucine into their corresponding alpha-ketoacids. As alpha-amino group acceptor alpha-keto-beta-methylvaleric acid and alpha-ketoisovaleric acid was much more efficient than alpha-ketoglutarate. The sensorially and metabolic key intermediate on the pathway to the branched-chain fatty acids, 3-methylbutanoic acid was produced from leucine at the onset of the stationary growth phase and then, when the growth medium became scarce in leucine, from the oxidation of glucose via pyruvate. CONCLUSIONS: This paper demonstrates that the sensorially important branched-chain aldehydes and acids are important intermediates on the metabolic route leading to branched-chain fatty acids for cell membrane biosynthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic information obtained is extremely important in connection with a future biotechnological design of starter cultures for production of fermented meat.     

6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering

The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. In vitro studies have previously demonstrated that the loading module of 6-deoxyerythronolide B synthase (DEBS) exhibits relaxed substrate specificity and is able to accept butyryl-CoA, leading to the production of polyketides with butyrate starter units. We have shown that we can produce butyryl-CoA at levels of up to 50% of the total CoA pool in Escherichia coli cells that overexpress the acetoacetyl-CoA:acetyl-CoA transferase, AtoAD (EC 2.8.3.8), in media supplemented with butyrate. The DEBS polyketide synthase (PKS) used butyryl-CoA and methylmalonyl-CoA supplied in vivo by the AtoAD and methylmalonyl-CoA mutase pathways, respectively, to produce 15-methyl-6-dEB. Priming DEBS with endogenous butyryl-CoA affords an alternative and more direct route to 15-Me-6-dEB than that provided by the chemobiosynthesis method [Jacobsen, J. R., et al. (1997) Science 277, 367-369], which relies on priming a mutant DEBS with an exogenously fed diketide thioester. The approach described here demonstrates the utility of metabolic engineering in E. coli to introduce precursor pathways for the production of novel polyketides.     

Enzymes involved in fatty acid and polyketide biosynthesis in Streptomyces glaucescens: role of FabH and FabD and their acyl carrier protein specificity

Malonyl acyl carrier protein (ACP) is used as an extender unit in each of the elongation steps catalyzed by the type II dissociated fatty acid synthase (FAS) and polyketide synthase (PKS) of Streptomyces glaucescens. Initiation of straight-chain fatty acid biosynthesis by the type II FAS involves a direct condensation of acetyl-CoA with this malonyl-ACP to generate a 3-ketobutyryl-ACP product and is catalyzed by FabH. In vitro experiments with a reconstituted type II PKS system in the absence of FabH have previously shown that the acetyl-ACP (generated by decarboxylation of malonyl-ACP), not acetyl-CoA, is used to initiate tetracenomycin C (TCM C) biosynthesis. We have shown that sgFabH activity is present in S. glaucescens fermentations during TCM C production, suggesting that it could contribute to initiation of TCM C biosynthesis in vivo. Isotope incorporation studies with [CD3]acetate and [13CD3]acetate demonstrated significant intact retention of three deuteriums into the starter unit of palmitate and complete washout of deuterium label into the starter unit of TCM C. These observations provide evidence that acetyl-CoA is not used directly as a starter unit for TCM C biosynthesis in vivo and argue against an involvement of FabH in this process. Consistent with this conclusion, assays of the purified recombinant sgFabH with acetyl-CoA demonstrated activity using malonyl-ACP generated from either FabC (the S. glaucescens FAS ACP) (k(cat) 42.2 min(-1), K(m) 4.5 +/- 0.3 microM) or AcpP (the E. coli FAS ACP) (k(cat) 7.5 min(-1), K(m) 6.3 +/- 0.3 microM) but not TcmM (the S. glaucescens PKS ACP). In contrast, the sgFabD which catalyzes conversion of malonyl-CoA to malonyl-ACP for fatty acid biosynthesis was shown to be active with TcmM (k(cat) 150 min(-1), K(m) 12.2 +/- 1.2 microM), AcpP (k(cat) 141 min(-1), K(m) 13.2 +/- 1.6 microM), and FabC (k(cat) 560 min(-1), K(m) 12.7 +/- 2.6 microM). This enzyme was shown to be present during TCM C production and could play a role in generating malonyl-ACP for both processes. Previous demonstrations that the purified PKS ACPs catalyze self-malonylation and that a FabD activity is not required for polyketide biosynthesis are shown to be an artifact of the expression and purification protocols. The relaxed ACP specificity of FabD and the lack of a clear alternative are consistent with a role of FabD in providing malonyl-ACP precursors for PKS as well as FAS processes. In contrast, the ACP specificity of FabH, isotope labeling studies, and a demonstrated alternative mechanism for initiation of the PKS process provide unequivocal evidence that FabH is involved only in the FAS process.     

微生物源脂肽的生物合成和分子调控

微生物源脂肽具有抑制真菌和细菌的生长、抗病毒和抗肿瘤等多种生物活性,在农业生物防治、临床医疗、环境治理等多种领域具有巨大的应用潜力。然而,低产量一直是影响其推广应用的瓶颈。深入了解脂肽合成的关键因素和调控策略对于提高其产量和纯度至关重要。本文概括了3大家族脂肽surfactin、fengycin和iturin的结构、功能及应用前景,介绍了NRPS和NRPS-PKS两种合成系统的结构域和功能,阐释了脂肽生物合成过程中侧链脂肪酸的合成、脂肪酸的活化及与氨基酸的连接、肽链的延伸和环化三个阶段的模块组装和酶催化活动,以及三大家族脂肽合成操纵子开放阅读框的组成;总结了导入或缺失关键基因、定点突变、模块替换、强启动子替换、修饰前体路径等多种遗传操作对脂肽产量的影响,以及群体感应肽信息素、sigma因子等全局调控因子对脂肽合成基因表达的调节。指出利用多组学联用深入探讨脂肽合成的全局分子调控机制和加强结构域蛋白互作和分子动力学研究是提高脂肽产量和纯度以及创造新脂肽的理论基础,提出了利用基因组装和编辑等合成生物学方法及代谢工程技术提高脂肽产量和挖掘新型脂肽靶向性的可能途径,为推进脂肽的生产和应用进程提供科学参考。     

Biotechnological production of eicosapentaenoic acid: From a metabolic engineering point of view

Eicosapentaenoic acid (EPA) is an ω3 polyunsaturated fatty acid which has been demonstrated to play important roles in a number of aspects of human health. EPA is traditionally obtained from marine fish oils. However, the shrinking fish populations are making the sustainability of these sources questionable. Consequently, alternative sources of EPA are being sought, especially from marine microalgae, bacteria, and fungi. These microorganisms contain relatively large amounts of high-quality EPA and they are the primary producers of this important fatty acid. There are two distinct pathways for EPA de novo biosynthesis in microbial systems: the desaturation and elongation pathway and the polyketide pathway. Genes involved in the biosynthetic pathways have been identified from different microorganisms and characterized in depth. In addition, numerous strategies have been developed for commercial production of EPA by microbial fermentation, among which strain improvements by genetic engineering could provide high-yield producers of EPA. In this review, we summarize recent efforts and experiences devoted to metabolic engineering of various microorganisms that lead to efficient biocatalysts for the production of EPA, as well as the key limitations and challenges. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of EPA biosynthesis and a greater potential of microbial production. Continued advances in metabolic engineering will help to improve the final titer, productivity, and yield of EPA.     

Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.     

Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals

Research towards the commercialization of fungible biofuels has received a great deal of recent interest and investment. To this end the microbial production of fatty acid-derived fuels from sustainable feedstocks is emerging as a viable option with rapid advances from both industry and academia. The manipulation of the fatty acid biosynthesis pathway, especially in Escherichia coli, has been widely studied and several approaches that increase fatty acid production have been identified. However, further advances will be required for the economic large-scale production of fatty acid-derived biofuels. Here we present an overview of fatty acid biosynthesis and its regulation in E. coli from a metabolic engineering viewpoint and offer potential approaches and considerations for the microbial overproduction of custom designed fatty acids for use as biofuels or in the manufacture of oleochemicals.