Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae

3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l⁻¹. Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future.     

Evaluation of 3-hydroxypropionate biosynthesis in vitro by partial introduction of the 3-hydroxypropionate/4-hydroxybutyrate cycle from <Emphasis Type="Italic">Metallosphaera sedula</Emphasis>

The chemical 3-hydroxypropionate (3HP) is an important starting reagent for the commercial synthesis of specialty chemicals. In this study, a part of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula was utilized for 3HP production. To study the basic biochemistry of this pathway, an in vitro-reconstituted system was established using acetyl-CoA as the substrate for the kinetic analysis of this system. The results indicated that 3HP formation was sensitive to acetyl-CoA carboxylase and malonyl-CoA reductase, but not malonate semialdehyde reductase. Also, the competition between 3HP formation and fatty acid production was analyzed both in vitro and in vivo. This study has highlighted how metabolic flux is controlled by different catalytic components. We believe that this reconstituted system would be valuable for understanding 3HP biosynthesis pathway and for future engineering studies to enhance 3HP production.     

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.     

Production of the plant polyketide curcumin in Aspergillus oryzae: strengthening malonyl-CoA supply for yield improvement

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae. Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 μg/plate) on an agar medium containing feruloyl-N-acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus. This study provided useful information for heterologous polyketide production in A. oryzae.     

Anaerobic degradation of malonatevia malonyl-CoA bySporomusa malonica,Klebsiella oxytoca,andRhodobacter capsulatus

Anaerobic decarboxylation of malonate to acetate was studied withSporomusa malonica, Klebsiella oxytoca, andRhodobacter capsulatus. WhereasS. malonica could grow with malonate as sole substrate (Y=2.0 g·mol^–1), malonate decarboxylation byK. oxytoca was coupled with anaerobic growth only in the presence of a cosubstrate, e.g. sucrose or yeast extract (Y _s =1.1–1.8 g·mol malonate^–1).R. capsulatus used malonate anaerobically only in the light, and growth yields with acetate and malonate were identical. Malonate decarboxylation in cell-free extracts of all three bacteria was stimulated by catalytic amounts of malonyl-CoA, acetyl-CoA, or Coenzyme A plus ATP, indicating that actually malonyl-CoA was the substrate of decarboxylation. Less than 5% of malonyl-CoA decarboxylase activity was found associated with the cytoplasmic membrane. Avidin (except forK. oxytoca) and hydroxylamine inhibited the enzyme completely, EDTA inhibited partially. InS. malonica andK. oxytoca, malonyl-CoA decarboxylase was active only after growth with malonate; malonyl-CoA: acetate CoA transferase was found as well. These results indicate that malonate fermentation by these bacteria proceedsvia malonyl-CoA mediated by a CoA transferase and that subsequent decarboxylation to acetyl-CoA is catalyzed, at least withS. malonica andR. capsulatus, by a biotin enzyme.Abbreviations CoASH Coenzyme A - EDTA ethylenediamine tetraacetate     

Metabolic engineering of type II methanotroph,Methylosinus trichosporium OB3b,for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway

We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP⁺-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.     

Biosynthetic pathway for poly(3-Hydroxypropionate) in recombinant Escherichia coli

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.     

Carnitine palmitoyltransferase in the heart is controlled by a different mechanism than the hepatic enzyme

Diminished sensitivity of hepatic carnitine palmitoyltransferase to inhibition by malonyl-CoA in the fasting and diabetic states is a well-recognized aspect of the regulatory mechanism forhepatic fatty acid oxidation. Inhibition of myocardial carnitine palmitoyltransferase by malonyl-CoA may play an important role in regulation of fatty acid oxidation in the heart, but there has been a discrepancy in data relating to changes in malonyl-CoA sensitivity of the myocardial carnitine palmitoyltransferase during fasting. Analysis of malonyl-CoA inhibition of myocardial carnitine palmitoyltransferase in fasting and fed states under a variety of conditions has indicated that under no condition could any difference be found in malonyl-CoA sensitivity that was attributable to fasting. Proteolysis of the outer carnitine palmitoyltransferase led to artifactual changes in sensitivity due to the appearance of partial inhibition. We have concluded that the sensitivity of myocardial carnitine palmitoyltransferase to malonyl-CoA does not change during fasting. Changes in fatty acid oxidation in the heart are probably due to changes in malonyl-CoA concentrations or to other inhibitors. (Mol Cell Biochem 116: 39–45, 1992)     

Isolation and Structures of New Peptide Antibiotics,Cirratiomycin A and B

We measured the concentrations of acetyl-CoA and malonyl-CoA in shoots and roots of corn (Zea mays, L., cv. “Peter Corn”). Acetyl-CoA and malonyl-CoA concentrations were found to be relatively constant in shoots and in roots under a light-dark cycle. Acetyl-CoA concentrations were lower in shoots than in roots, whereas malonyl-CoA concentrations were higher in shoots than in roots.     

Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol

3‐Hydroxypropionic acid (3‐HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3‐HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an “imbalance between the two enzymes” and the “instability of the first enzyme DhaB” were the major factors limiting 3‐HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl‐thio‐β‐galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), α‐ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH‐BGK1 showed the highest level of 3‐HP production (2.8 g/L) under shake‐flask conditions. When an aerobic fed‐batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH‐BGK1 produced 38.7 g 3‐HP/L with an average yield of 35%. This article reports the highest level of 3‐HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729–739 © 2009 Wiley Periodicals, Inc.     

Discovery and analysis of novel metabolic pathways for the biosynthesis of industrial chemicals: 3‐hydroxypropanoate

Sustainable microbial production of high‐value organic compounds such as 3‐hydroxypropanoate (3HP) is becoming an increasingly attractive alternative to organic syntheses that utilize petrochemical feedstocks. We applied the Biochemical Network Integrated Computational Explorer (BNICE) framework to the automated design and evaluation of novel biosynthetic routes for the production of 3HP from pyruvate. Among the pathways generated by the BNICE framework were all of the known pathways for the production of 3HP as well as numerous novel pathways. The pathways generated by BNICE were ranked based on four criteria: pathway length, thermodynamic feasibility, maximum achievable yield to 3HP from glucose, and maximum achievable activity at which 3HP can be produced. Four pathways emerged from this ranking as the most promising for the biosynthesis of 3HP, and three of these pathways, including the shortest pathways discovered, were novel. We also discovered novel routes for the biosynthesis of 28 commercially available compounds that are currently produced exclusively through organic synthesis. Examination of the optimal pathways for the biosynthesis of these 28 compounds in E. coli revealed pyruvate and succinate to be ideal intermediates for achieving high product yields from glucose. Biotechnol. Bioeng. 2010; 106: 462–473. © 2010 Wiley Periodicals, Inc.     

Radiochemical malonyl-CoA decarboxylase assay: activity and subcellular distribution in heart and skeletal muscle

Malonyl-CoA decarboxylase is the main route for the disposal of malonyl-CoA, the key metabolite in the regulation of mitochondrial fatty acid oxidation. We have developed a simple and sensitive radiochemical assay to determine malonyl-CoA decarboxylase activity. The decarboxylation of [2-14C]malonyl-CoA produces [2-14C]acetyl-CoA, which is converted to [2-14C]acetylcarnitine in the presence of excess L-carnitine and carnitine acetyltransferase. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate and the radioactivity measured by scintillation counting. Measurement of malonyl-CoA decarboxylase activities with this method gives values comparable to those obtained with assays currently in use, but has the advantage of being simpler and less labor intensive. We have applied this assay to rat skeletal muscle of different fiber-type composition and to rat heart. Malonyl-CoA decarboxylase activity (mU/g wet wt) correlates with the oxidative capacity of the muscles, being lowest in type IIb fibers (42.7 +/- 3.0) and highest in heart (1071.4 +/- 260), with intermediate activity in type IIa fibers (150.7 +/- 4.3) and type I fibers (107.8 +/- 7.6). Studies on subcellular distribution of malonyl-CoA decarboxylase activity in rat heart and rat skeletal muscle show that approximately 50 and 65% is localized to mitochondria, while 50 and 35% of the activity is extramitochondrial.     

Modulation of the central carbon metabolism of Corynebacterium glutamicum improves malonyl-CoA availability and increases plant polyphenol synthesis

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L ⁻¹ (0.088 mM) naringenin or 112 mg·L ⁻¹ (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis.     

Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO₂ directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L⁻¹ (348.8 mg/g dry cell weight) 3-HP directly from CO₂ in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO₂ in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO₂ fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.     

Characterization of overt carnitine palmitoyltransferase in rat platelets; involvement of insulin on its regulation

Summary Saponin-permeabilization (30 µg/ml) of the platelet plasma membrane, which enables access of added compounds to mitochondrial overt carnitine palmitoyltransferase (CPT I), was applied to allow the rapid determination of CPT I activity in situ. The effects of diabetes and short-term incubation with insulin in vitro on the kinetic parameters and malonyl-CoA sensitivity of CPT I were also studied in rat platelets. CPT I exhibited ordinary Michaelis-Menten kinetics when platelets were incubated with palmitoyl-CoA. Malonyl-CoA showed an I₅₀ (concentration giving 50% inhibition of CPT activity) of 0.92 ± 0.11 µM in permeabilized platelets. Platelets obtained from diabetic rats (induced by streptozotocin injection) exhibited an increased V_max. and I₅₀ for malonyl-CoA, and an unaltered K_m for palmitoyl-CoA. In contrast, preincubation of platelets prepared from both fed control rats and diabetic rats with insulin (100 and 150 µ-cU/ml) led to a decrease in enzyme activity when assayed with 75 µM palmitoyl-CoA and 0.5 mM L-carnitine as substrates. These in vivo and in vitro results suggested that insulin directly modulated rat platelet CPT I activity, as it does in the liver.     

Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO₂ in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase). The model correctly predicted previously observed features of the cycle: the 35–65% split of carbon flux through the acetyl-CoA and succinate branches, the high abundance and relative ratio of acetyl-CoA/propionyl-CoA carboxylase (ACC) and MCR, and the significance of ACC and hydroxybutyryl-CoA synthetase (HBCS) as regulated control points for the cycle. The model was then used to assess metabolic engineering strategies for incorporating CO₂ into chemical intermediates and products of biotechnological importance: acetyl-CoA, succinate, and 3-hydroxypropionate.     

Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria

Direct conversion of carbon dioxide into chemicals using engineered autotrophic microorganisms offers a potential solution for both sustainability and carbon mitigation. Butyrate is an important chemical used in various industries, including fragrance, food, and plastics. A model cyanobacterium Synechococcus elongatus PCC 7942 was engineered for the direct photosynthetic conversion of CO ₂ to butyrate. An engineered Clostridium Coenzyme A (CoA)-dependent pathway leading to the synthesis of butyryl-CoA, the precursor to butyrate, was introduced into S. elongatus PCC 7942. Two CoA removal strategies were then individually coupled to the modified CoA-dependent pathway to yield butyrate production. Similar results were observed between the two CoA removal strategies. The best butyrate producing strain of S. elongatus resulted in an observed butyrate titer of 750 mg/L and a cumulative titer of 1.1 g/L. These results demonstrated the feasibility of photosynthetic butyrate production and expanded the chemical repertoire accessible for production by photoautotrophs.     

Determination of Acetyl-CoA and Malonyl-CoA in Germinating Rice Seeds Using the LC-MS/MS Technique

A highly sensitive and selective analytical method was developed to determine levels of acetyl-CoA and malonyl-CoA in plant tissues. The analytical method includes a convenient extraction method for plant samples and a new LC-MS/MS technique utilizing an ion pair reagent. These acyl-CoAs, present in germinating rice seeds, were then determined by the method developed. It was found that the concentrations of both acetyl-CoA and malonyl-CoA increased with time during the germination of rice seeds and also increased at the elevated cultivation temperatures among tested. These results reflect the development of enzymes that produce these acyl-CoAs in germinating rice seeds.     

Chloroflexus aurantiacus secretes 3-hydroxypropionate,a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 M). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO₂ fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using ¹³C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO₂. The 3HP formed by cells grown on propionate and ¹³CO₂ was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO₂ fixation.Abbreviations 3HP 3-hydroxypropionate - 2F3HP 2,fluoro,3-hydroxypropionate - FAc fluoroacetate - GC gas chromatography - MS mass spectrometry - NMR nuclear magnetic resonance     

Hypothalamic malonyl-coenzyme A and the control of energy balance

An intermediate in the fatty acid biosynthetic pathway, malonyl-coenzyme A (CoA), has emerged as a major regulator of energy homeostasis not only in peripheral metabolic tissues but also in regions of the central nervous system that control satiety and energy expenditure. Fluctuations in hypothalamic malonyl-CoA lead to changes in food intake and peripheral energy expenditure in a manner consistent with an anorexigenic signaling intermediate. Hypothalamic malonyl-CoA is regulated by nutritional and endocrine cues including glucose and leptin, respectively. That malonyl-CoA is an essential component in the energy homeostatic signaling system of the hypothalamus is supported by convergence of physiological, pharmacological, and genetic evidence. This review will focus on evidence implicating malonyl-CoA as a central player in the control of body weight and adiposity as well as clues to the molecular mechanism by which carbon flux through the fatty acid biosynthetic pathway is linked to the neural control of energy balance.