Discovery of novel pathways for carbohydrate metabolism

Closing the gap between the increasing availability of complete genome sequences and the discovery of novel enzymes in novel metabolic pathways is a significant challenge. Here, we review recent examples of assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized proteins, with a focus on enzymes and metabolic pathways involved in the catabolism and biosynthesis of monosaccharides and polysaccharides. The most effective approaches are based on analyses of sequence-function space in protein families that provide clues for the predictions of the functions of the uncharacterized enzymes. As summarized in this Opinion, this approach allows the discovery of the catabolism of new molecules, new pathways for common molecules, and new enzymatic chemistries.     

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.     

The methanolic extract of Garcinia atroviridis (MeGa) reduces body weight and food intake,and improves lipid profiles by altering the lipid metabolism: a rat model

Garcinia species are widely used for their slimming effects via increased fat burning and suppression of satiety. However, scientific evidence for the biological effects of Garcinia atroviridis (GA) is lacking. We investigated the phytochemical composition, safety profiles, and antioxidant and antiobesity effects of methanolic extracts of Garcinia atroviridis (MeGa) in obese female rats. Repeated dose toxicity studies were conducted according to the OECD guidelines. Upon sacrifice, haematological, biochemical, lipid profile, and serum-based metabolomics analyses were performed to evaluate metabolic expression changes and their related pathways. MeGa contains several phytochemical groups and GA fruit acids. MeGa was found to be nontoxic in both male and female rats with an oral lethal dose (LD50) of 2000 mg/kg. After 9 weeks of treatment, MeGa-treated obese rats had lower weight gain and better lipid profiles (cholesterol and triglyceride), which correlated with the altered metabolic pathways involved in the metabolism of lipid (glycerophospholipid) and biosynthesis of unsaturated fatty acid. In addition, MeGa caused differential metabolism pathways of arachidonic acid and tryptophan that affect the inflammatory response and suppression of appetite. We concluded that MeGa is safe, and its slimming effects are due to the differential metabolism of lipids.     

Improved production of long-chain fatty acid in Escherichia coli by an engineering elongation cycle during fatty acid synthesis (FAS) through genetic manipulation

The microbial biosynthesis of fatty acid of lipid metabolism, which can be used as precursors for the production of fuels of chemicals from renewable carbon sources, has attracted significant attention in recent years. The regulation of fatty acid biosynthesis pathways has been mainly studied in a model prokaryote, Escherichia coli. During the recent period, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis, as well as in Streptococcus pneumonia. The goal of this study was to increase the production of long-chain fatty acids by developing recombinant E. coli strains that were improved by an elongation cycle of fatty acid synthesis (FAS). The fabB, fabG, fabZ, and fabI genes, all homologous of E. coli, were induced to improve the enzymatic activities for the purpose of overexpressing components of the elongation cycle in the FAS pathway through metabolic engineering. The beta-oxoacyl-ACP synthase enzyme catalyzed the addition of acyl-ACP to malonyl-ACP to generate beta- oxoacyl-ACP. The enzyme encoded by the fabG gene converted beta-oxoacyl-ACP to beta-hydroxyacyl-ACP, the fabZ catalyzed the dehydration of beta-3-hydroxyacyl-ACP to trans-2-acyl-ACP, and the fabI gene converted trans-2- acyl-ACP to acyl-ACP for long-chain fatty acids. In vivo productivity of total lipids and fatty acids was analyzed to confirm the changes and effects of the inserted genes in E. coli. As a result, lipid was increased 2.16-fold higher and hexadecanoic acid was produced 2.77-fold higher in E. coli JES1030, one of the developed recombinants through this study, than those from the wild-type E. coli.     

Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi

Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.     

Engineered biosynthesis of medium-chain esters in Escherichia coli

Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in Escherichia coli for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in E. coli were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from Saccharomyces cerevisiae was the best enzyme for the formation of both IBAc and IAAc in E. coli. In vitro biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δldh, ΔpoxB, Δpta) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36 g/L of IBAc was produced after 72 h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.     

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO₂-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.     

Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids

One of the most promising alternatives to petroleum for the production of fuels and chemicals is bio-oil based chemistry. Microbial oils are gaining importance because they can be engineered to accumulate lipids enriched in desired fatty acids. These specific lipids are closer to the commercialized product, therefore reducing pollutants and costly chemical steps. Yarrowia lipolytica is the most widely studied and engineered oleaginous yeast. Different molecular and bioinformatics tools permit systems metabolic engineering strategies in this yeast, which can produce usual and unusual fatty acids. Usual fatty acids, those usually found in triacylglycerol, accumulate through the action of several pathways, such as fatty acid/triacylglycerol synthesis, transport and degradation. Unusual fatty acids are enzymatic modifications of usual fatty acids to produce compounds that are not naturally synthetized in the host. Recently, the metabolic engineering of microorganisms has produced different unusual fatty acids, such as building block ricinoleic acid and nutraceuticals such as conjugated linoleic acid or polyunsaturated fatty acids. Additionally, microbial sources are preferred hosts for the production of fatty acid-derived compounds such as γ-decalactone, hexanal and dicarboxylic acids. The variety of lipids produced by oleaginous microorganisms is expected to rise in the coming years to cope with the increasing demand.     

Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Pseudomonas putida is evolutionarily endowed with features relevant for bioproduction, especially under harsh operating conditions. The rich metabolic versatility of this species, however, comes at the price of limited formation of acetyl-coenzyme A (CoA) from sugar substrates. Since acetyl-CoA is a key metabolic precursor for a number of added-value products, in this work we deployed an in silico-guided rewiring program of central carbon metabolism for upgrading P. putida as a host for acetyl-CoA–dependent bioproduction. An updated kinetic model, integrating fluxomics and metabolomics datasets in addition to manually-curated information of enzyme mechanisms, identified targets that would lead to increased acetyl-CoA levels. Based on these predictions, a set of plasmids based on clustered regularly interspaced short palindromic repeats (CRISPR) and dead CRISPR-associated protein 9 (dCas9) was constructed to silence genes by CRISPR interference (CRISPRi). Dynamic reduction of gene expression of two key targets (gltA, encoding citrate synthase, and the essential accA gene, encoding subunit A of the acetyl-CoA carboxylase complex) mediated an 8-fold increase in the acetyl-CoA content of rewired P. putida. Poly(3-hydroxybutyrate) (PHB) was adopted as a proxy of acetyl-CoA availability, and two synthetic pathways were engineered for biopolymer accumulation. By including cell morphology as an extra target for the CRISPRi approach, fully rewired P. putida strains programmed for PHB accumulation had a 5-fold increase in PHB titers in bioreactor cultures using glucose. Thus, the strategy described herein allowed for rationally redirecting metabolic fluxes in P. putida from central metabolism towards product biosynthesis—especially relevant when deletion of essential pathways is not an option.     

Autophagy is required for lipid homeostasis during dark-induced senescence

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

Autophagy is required for the mobilization of membrane lipid components and lipid droplet dynamics during extended darkness in Arabidopsis.     

Thiamine in plants: Aspects of its metabolism and functions

Thiamine diphosphate (vitamin B₁) plays a fundamental role as an enzymatic cofactor in universal metabolic pathways including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. In addition, thiamine diphosphate has recently been shown to have functions other than as a cofactor in response to abiotic and biotic stress in plants. Recently, several steps of the plant thiamine biosynthetic pathway have been characterized, and a mechanism of feedback regulation of thiamine biosynthesis via riboswitch has been unraveled. This review focuses on these most recent advances made in our understanding of thiamine metabolism and functions in plants. Phenotypes of plant mutants affected in thiamine biosynthesis are described, and genomics, proteomics, and metabolomics data that have increased further our knowledge of plant thiamine metabolic pathways and functions are summarized. Aspects of thiamine metabolism such as catabolism, salvage, and transport in plants are discussed.     

Yeast and cancer cells – common principles in lipid metabolism

One of the paradigms in cancer pathogenesis is the requirement of a cell to undergo transformation from respiration to aerobic glycolysis – the Warburg effect – to become malignant. The demands of a rapidly proliferating cell for carbon metabolites for the synthesis of biomass, energy and redox equivalents, are fundamentally different from the requirements of a differentiated, quiescent cell, but it remains open whether this metabolic switch is a cause or a consequence of malignant transformation. One of the major requirements is the synthesis of lipids for membrane formation to allow for cell proliferation, cell cycle progression and cytokinesis. Enzymes involved in lipid metabolism were indeed found to play a major role in cancer cell proliferation, and most of these enzymes are conserved in the yeast, Saccharomyces cerevisiae. Most notably, cancer cell physiology and metabolic fluxes are very similar to those in the fermenting and rapidly proliferating yeast. Both types of cells display highly active pathways for the synthesis of fatty acids and their incorporation into complex lipids, and imbalances in synthesis or turnover of lipids affect growth and viability of both yeast and cancer cells. Thus, understanding lipid metabolism in S. cerevisiae during cell cycle progression and cell proliferation may complement recent efforts to understand the importance and fundamental regulatory mechanisms of these pathways in cancer.     

Lipid metabolism and Drosophila sperm development

Lipids are essential membrane structural components and important signal carriers. The major enzymatic metabolisms of various lipids (phospholipid, sphingolipid, cholesterol) are well studied. The developmental function of lipid metabolism has remained, for the most part, elusive. With the help of new techniques and model organisms, the important roles of lipid metabolism in development just start to emerge. Drosophila spermatogenesis is an ideal system for in vivo studies of cytokinesis and membrane remodeling during development. The metabolic regulators of many lipids, including phosphatidylinositol (PI) lipids, fatty acids and cholesterol, are reported to play critical roles in various steps during Drosophila spermatogenesis. In this mini-review, we summarized recent findings supporting a tight link between lipids metabolism and Drosophila sperm development.     

A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery

Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.     

Specialized ER membrane domains for lipid metabolism and transport

The endoplasmic reticulum (ER) is a highly organized organelle that performs vital functions including de novo membrane lipid synthesis and transport. Accordingly, numerous lipid biosynthesis enzymes are localized in the ER membrane. However, it is now evident that lipid metabolism is sub-compartmentalized within the ER and that lipid biosynthetic enzymes engage with lipid transfer proteins (LTPs) to rapidly shuttle newly synthesized lipids from the ER to other organelles. As such, intimate relationships between lipid metabolism and lipid transfer pathways exist within the ER network. Notably, certain LTPs enhance the activities of lipid metabolizing enzymes; likewise, lipid metabolism can ensure the specificity of LTP transfer/exchange reactions. Yet, our understanding of these mutual relationships is still emerging. Here, we highlight past and recent key findings on specialized ER membrane domains involved in efficient lipid metabolism and transport and consider unresolved issues in the field.