The initiation ketosynthase (FabH) is the sole rate-limiting enzyme of the fatty acid synthase of Synechococcus sp. PCC 7002

Cyanobacteria are Gram-negative bacteria that are desirable hosts for biodiesel production, because they are photosynthetic, relatively fast growing, and can secrete products. We have reconstituted the fatty acid synthase (FAS) of the cyanobacterium Synechococcus sp. PCC 7002 and subjected it to in vitro kinetic analysis. Our data revealed that the overall rate of this metabolic pathway is exclusively limited by the FabH ketosynthase, which initiates product synthesis by condensing malonyl-ACP with acetyl-CoA to form acetoacetyl-ACP. This finding sharply contrasts with our previous findings that the Escherichia coli FAS is predominantly limited by its dehydratase (FabZ) and enoyl reductase (FabI) activities and that FabH activity is not limiting. We therefore reconstituted and analyzed a set of “hybrid” FASs. When the Synechococcus FabH was used to replace its counterpart in the reconstituted E. coli FAS, the resulting synthase was strongly limited by FabH activity. Conversely, replacement of the E. coli FabZ with its Synechococcus homolog dramatically alleviated the dependence of E. coli FAS activity on FabZ. In agreement with this finding, introduction of the E. coli FabH in the Synechococcus FAS virtually eliminated its dependence on this subunit, whereas substitution of the Synechococcus FabZ with its E. coli homolog shifted a substantial fraction of the overall flux control in the Synechococcus FAS to FabZ. Our findings demonstrate that the rate-limiting steps can differ dramatically between closely related bacterial fatty acid synthases, and that such regulatory behavior is fundamentally the property of the controlling enzyme(s).     

Metabolic engineering of fatty acyl-ACP reductase-dependent pathway to improve fatty alcohol production in Escherichia coli

Fatty alcohols are important components of surfactants and cosmetic products. The production of fatty alcohols from sustainable resources using microbial fermentation could reduce dependence on fossil fuels and greenhouse gas emission. However, the industrialization of this process has been hampered by the current low yield and productivity of this synthetic pathway. As a result of metabolic engineering strategies, an Escherichia coli mutant containing Synechococcus elongatus fatty acyl-ACP reductase showed improved yield and productivity. Proteomics analysis and in vitro enzymatic assays showed that endogenous E. coli AdhP is a major contributor to the reduction of fatty aldehydes to fatty alcohols. Both in vitro and in vivo results clearly demonstrated that the activity and expression level of fatty acyl-CoA/ACP reductase is the rate-limiting step in the current protocol. In 2.5-L fed-batch fermentation with glycerol as the only carbon source, the most productive E. coli mutant produced 0.75 g/L fatty alcohols (0.02 g fatty alcohol/g glycerol) with a productivity of up to 0.06 g/L/h. This investigation establishes a promising synthetic pathway for industrial microbial production of fatty alcohols.     

Kinetically guided,ratiometric tuning of fatty acid biosynthesis

Cellular metabolism is a nonlinear reaction network in which dynamic shifts in enzyme concentration help regulate the flux of carbon to different products. Despite the apparent simplicity of these biochemical adjustments, their influence on metabolite biosynthesis tends to be context-dependent, difficult to predict, and challenging to exploit in metabolic engineering. This study combines a detailed kinetic model with a systematic set of in vitro and in vivo analyses to explore the use of enzyme concentration as a control parameter in fatty acid synthesis, an essential metabolic process with important applications in oleochemical production. Compositional analyses of a modeled and experimentally reconstituted fatty acid synthase (FAS) from Escherichia coli indicate that the concentration ratio of two native enzymes—a promiscuous thioesterase and a ketoacyl synthase—can tune the average length of fatty acids, an important design objective of engineered pathways. The influence of this ratio is sensitive to the concentrations of other FAS components, which can narrow or expand the range of accessible chain lengths. Inside the cell, simple changes in enzyme concentration can enhance product-specific titers by as much as 125-fold and elicit shifts in overall product profiles that rival those of thioesterase mutants. This work develops a kinetically guided approach for using ratiometric adjustments in enzyme concentration to control the product profiles of FAS systems and, broadly, provides a detailed framework for understanding how coordinated shifts in enzyme concentration can afford tight control over the outputs of nonlinear metabolic pathways.     

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP⁺ dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169 mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989 mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.     

Development of Escherichia coli MG1655 strains to produce long chain fatty acids by engineering fatty acid synthesis (FAS) metabolism

The goal of this research was to develop recombinant Escherichia coli to improve fatty acid synthesis (FAS). Genes encoding acetyl-CoA carboxylase (accA, accB, accC), malonyl-CoA-[acyl-carrier-protein] transacylase (fabD), and acyl-acyl carrier protein thioesterase (EC 3.1.2.14 gene), which are all enzymes that catalyze key steps in the synthesis of fatty acids, were cloned and over-expressed in E. coli MG1655. The acetyl-CoA carboxylase (ACC) enzyme catalyzes the addition of CO₂ to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC 3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. All the genes except for the EC 3.1.2.14 gene were homologous to E. coli genes and were used to improve the enzymatic activities to over-express components of the FAS pathway through metabolic engineering. All recombinant E. coli MG1655 strains containing various gene combinations were developed using the pTrc99A expression vector. To observe changes in metabolism, the in vitro metabolites and fatty acids produced by the recombinants were analyzed. The fatty acids (C16) from recombinant strains were produced 1.23-2.41 times higher than that from the wild type.     

Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.     

Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways

Fatty acid short-chain esters (FASEs) are biodiesels that are renewable, nontoxic, and biodegradable biofuels. A novel approach for the biosynthesis of FASEs has been developed using metabolically-engineered E. coli through combination of the fatty acid and 2-keto acid pathways. Several genetic engineering strategies were also developed to increase fatty acyl-CoA availability to improve FASEs production. Fed-batch cultivation of the engineered E. coli resulted in a titer of 1008 mg/L FASEs. Since the fatty acid and 2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in a variety of microorganisms to produce various FASEs from cheap and readily-available, renewable, raw materials such as sugars and cellulose in the future.     

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and ‘tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.     

An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction

We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.     

Neutral Lipid Biosynthesis in Engineered Escherichia coli: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.     

Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.     

Conversion of coconut oil to methyl ketones by two Aspergillus species

Isolates of Aspergillus ruber and A. repens have been grown on coconut oil as the sole carbon source in shake culture. Methyl ketones (C₅-C₁₃) were isolated by solvent extraction and analysed by combined gas chromatography and mass spectrometry. 2-Undecanone was the main volatile product reflecting the high concentration of dodecanoic acid in the original coconut oil. The reactivity of the individual short chain fatty acids as substrates for production of methyl ketones would appear to decrease with increasing molecular weight of the acid after taking into account the greater volatility of the lower molecular weight homologues. 2-Hexanone and 2-octanone were produced by all isolates in low concentration (< 1%). Nonanoic acid and 2-heptanone were converted into 2-octanone. Low concentrations of secondary alcohols were formed under aerobic conditions. It is suggested that the production of methyl ketones by partial β-oxidation is too closely related to mainstream metabolism to be of use in the biochemical taxonomy of the genus.     

A high yield optimized method for the production of acylated ACPs enabling the analysis of enzymes involved in P. falciparum fatty acid biosynthesis

The natural substrates of the enzymes involved in type-II fatty acid biosynthesis (FAS-II) are acylated acyl carrier proteins (acyl-ACPs). The state of the art method to produce acyl-ACPs involves the transfer of a phosphopantetheine moiety from CoA to apo-ACP by E. coli holo-ACP synthase (EcACPS), yielding holo-ACP which subsequently becomes thioesterified with free fatty acids by the E. coli acyl-ACP synthase (EcAAS). Alternatively, acyl-ACPs can be synthesized by direct transfer of acylated phosphopantetheine moieties from acyl-CoA to apo-ACP by means of EcACPS. The need for native substrates to characterize the FAS-II enzymes of P. falciparum prompted us to investigate the potential and limit of the two methods to efficiently acylate P. falciparum ACP (PfACP) with respect to chain length and β-modification and in preparative amounts. The EcAAS activity is found to be independent from the oxidation state at the β-position and accepts fatty acids as substrates with chain lengths starting from C8 to C20, whereas EcACPS accepts very efficiently acyl-CoAs with chain lengths up to C16, and with decreasing activity also longer chains (C18 to C20). Methods were developed to synthesize and purify preparative amounts of high quality natural substrates that are fully functional for the enzymes of the P. falciparum FAS-II system.     

Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ∼35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.     

Ketonic rancidity in coconut due to xerophilic fungi

A type of deterioration called ketonic rancidity occurred in coconut after inoculation with four xerophilic fungi, Eurotium amstelodami, E. chevalieri, E. herbariorum and Penicillium citrinum. The fungi were incubated at low water activity and oxygen tension. A homologous series of aliphatic methyl ketones and secondary alcohols C₅C₁₁ were isolated and identified in the rancid samples after fungal growth. Evidence is presented that odd numbered methyl ketones (C₅C₁₁) are derived from even numbered short chain fatty acids with one more carbon atoms than the ketones via a modified β-oxidation of the parent fatty acid. Heptan-2-one and heptan-2-ol are the main reaction products except in the case of E. herbariorum where even numbered hexan-2-one, hexan-2-ol, octan-2-one and octan-2-ol were produced. Other moulds grown on coconut under similar conditions—Aspergillus flavus Link and Chrysosporium farinicola (Burnside) Skou (C. fastidium Pitt)—did not cause ketonic deterioration.     

Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans

Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l^?1 linoleic acid, 23 g l^?1 cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l^?1 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l^?1 h^?1. This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.     

Improvement of fatty acid biosynthesis by engineered recombinant Escherichia coli

The purpose of this research was to develop new strains of Escherichia coli with improved fatty acid biosynthesis. β-Ketoacyl acyl carrier protein synthase III (fabH) catalyzes the first step in the synthesis of fatty acids in parallel with acetyl-CoA carboxylase (accABC) and malonyl-CoA: acyl carrier protein transacylase (fabD) in Escherichia coli K-12 MG1655. The enzyme encoded by the fabH gene leads to an increase in the synthesis of short-chain-length fatty acids and a strong preference for acetyl-CoA, as it produces only straight chain fatty acids (SCFAs). It also seems to play a role in determining the type and composition of fatty acids produced. In this study, metabolically engineered strains of E. coli K-12 MG1655 containing fabH or accA::accBC::fabD or accA::accBC:: fabD::fabH gene-inserted expression vector (pTrc99A) were constructed. To observe the effects of overexpression, the production of malonic acid, a pathway intermediate, and fatty acids was analyzed. The resulting recombinant strains produced total lipids up to approximately 1.2 ～ 1.6 fold higher than that of wild-type E. coli. The production of hexadecanoic acid was especially enhanced up to approximately 4.8 fold in E. coli SGJS13 as compared to E. coli SGJS11.     

Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies

We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of β-oxidation designed to overproduce β-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene. Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert β-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.