Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase.

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, ß-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and ß-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the ß-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.

Metabolic engineering of microorganisms for the production of structurally diverse esters.

Conventional petroleum-based chemical industry, although economically still thriving, is now facing great socio-political challenges due to the increasing concerns on climate change and limited availability of fossil resources. In this context, microbial production of fuels and commodity oleochemicals from renewable biomass is being considered a promising sustainable alternative. The increasing understanding of cellular systems has enabled the redesign of microbial metabolism for the production of compounds present in many daily consumer products such as esters, waxes, fatty acids (FA) and fatty alcohols. Small aliphatic esters are important flavour and fragrance elements while long-chain esters, composed of FA esterified to fatty alcohols, are widely used in lubricant formulas, paints, coatings and cosmetics. Here, we review recent advances in the biosynthesis of these types of mono alkyl esters in vivo. We focus on the critical ester bond-forming enzymes and the latest metabolic engineering strategies employed for the biosynthesis of a wide range of products ranging from low-molecular-weight esters to waxy compounds.

High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties.

BACKGROUND: Microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures. RESULTS: With the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 ± 5.3 mg MBE L-1 in batch cultures. Fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 ± 6.9 mg MBE L-1 with a volumetric productivity of 15.8 ± 1.1 mg MBE (L h)-1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies. CONCLUSIONS: The analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.

Overexpression of a phosphatidic acid phosphatase type 2 leads to an increase in triacylglycerol production in oleaginous Rhodococcus strains.

Oleaginous Rhodococcus strains are able to accumulate large amounts of triacylglycerol (TAG). Phosphatidic acid phosphatase (PAP) enzyme catalyzes the dephosphorylation of phosphatidic acid (PA) to yield diacylglycerol (DAG), a key precursor for TAG biosynthesis. Studies to establish its role in lipid metabolism have been mainly focused in eukaryotes but not in bacteria. In this work, we identified and characterized a putative PAP type 2 (PAP2) encoded by the ro00075 gene in Rhodococcus jostii RHA1. Heterologous expression of ro00075 in Escherichia coli resulted in a fourfold increase in PAP activity and twofold in DAG content. The conditional deletion of ro00075 in RHA1 led to a decrease in the content of DAG and TAG, whereas its overexpression in both RHA1 and Rhodococcus opacus PD630 promoted an increase up to 10 to 15 % by cellular dry weight in TAG content. On the other hand, expression of ro00075 in the non-oleaginous strain Rhodococcus fascians F7 promoted an increase in total fatty acid content up to 7 % at the expense of free fatty acid (FFA), DAG, and TAG fractions. Moreover, co-expression of ro00075/atf2 genes resulted in a fourfold increase in total fatty acid content by a further increase of the FFA and TAG fractions. The results of this study suggest that ro00075 encodes for a PAP2 enzyme actively involved in TAG biosynthesis. Overexpression of this gene, as single one or with an atf gene, provides an alternative approach to increase the biosynthesis and accumulation of bacterial oils as a potential source of raw material for biofuel production.

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate.

Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production.

BACKGROUND: Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations. RESULTS: The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppß, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lppß genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the ß-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPß rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L(-1) at the end of the process. CONCLUSIONS: This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor.

BACKGROUND: Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. RESULTS: We have identified two S. coelicolor genes, named lppα (SCO1102) and lppß (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppß genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppß had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppß in the wild type strain of S. coelicolor led to a significant increase in TAG production. CONCLUSIONS: The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels production.

Emerging engineering principles for yield improvement in microbial cell design.

Metabolic Engineering has undertaken a rapid transformation in the last ten years making real progress towards the production of a wide range of molecules and fine chemicals using a designed cellular host. However, the maximization of product yields through pathway optimization is a constant and central challenge of this field. Traditional methods used to improve the production of target compounds from engineered biosynthetic pathways in non-native hosts include: codon usage optimization, elimination of the accumulation of toxic intermediates or byproducts, enhanced production of rate-limiting enzymes, selection of appropriate promoter and ribosome binding sites, application of directed evolution of enzymes, and chassis re-circuit. Overall, these approaches tend to be specific for each engineering project rather than a systematic practice based on a more generalizable strategy. In this mini-review, we highlight some novel and extensive approaches and tools intended to address the improvement of a target product formation, founded in sophisticated principles such as dynamic control, pathway genes modularization, and flux modeling.

FasR, a novel class of transcriptional regulator, governs the activation of fatty acid biosynthesis genes in Streptomyces coelicolor.

Membrane lipid homeostasis is essential for bacterial survival and adaptation to different environments. The regulation of fatty acid biosynthesis is therefore crucial for maintaining the correct composition and biophysical properties of cell membranes. This regulation implicates a biochemical control of key enzymes and a transcriptional regulation of genes involved in lipid metabolism. In Streptomyces coelicolor we found that control of lipid homeostasis is accomplished, at least in part, through the transcriptional regulation of fatty acid biosynthetic genes. A novel transcription factor, FasR (SCO2386), controls expression of fabDHPF operon and lies immediately upstream of fabD, in a cluster of genes that is highly conserved within actinomycetes. Disruption of fasR resulted in a mutant strain, with severe growth defects and a delay in the timing of morphological and physiological differentiation. Expression of fab genes was downregulated in the fasR mutant, indicating a role for this transcription factor as an activator. Consequently, the mutant showed a significant drop in fatty acid synthase activity and triacylglyceride accumulation. FasR binds specifically to a DNA sequence containing fabDHPF promoter region, both in vivo and in vitro. These data provide the first example of positive regulation of genes encoding core proteins of saturated fatty acid synthase complex.