Cytosolic Acetyl-CoA Generated by ATP-Citrate Lyase Is Essential for Acetylation of Cell Wall Polysaccharides.

Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.

Construction of artificial micro-aerobic metabolism for energy- and carbon-efficient synthesis of medium chain fatty acids in Escherichia coli.

Medium-chain (C6-C10) chemicals are important components of fuels, commodities and fine chemicals. Numerous exciting achievements have proven reversed ß-oxidation cycle as a promising platform to synthesize these chemicals. However, under native central carbon metabolism, energetic and redox constraints limit the efficient operation of reversed ß-oxidation cycle. Current fermentative platform has to use different chemically and energetically inefficient ways for acetyl-CoA and NADH biosynthesis, respectively. The characteristics such as supplementation of additional acetate and formate or high ATP requirement makes this platform incompatible with large-scale production. Here, an artificial micro-aerobic metabolism for energy and carbon-efficient conversion of glycerol to MCFAs was constructed to present solutions towards these barriers. After evaluating numerous bacteria pathways under micro-aerobic conditions, one synthetic metabolic step enabling biosynthesis of acetyl-CoA and NADH simultaneously, without any energy cost and additional carbon requirement, and reducing loss of carbon to carbon dioxide-emitting reactions, was conceived and successfully constructed. The pyruvate dehydrogenase from Enterococcus faecalis was identified and biochemically characterized, demonstrating the most suitable characteristics. Furthermore, the carbon and energy metabolism in Escherichia coli was rewired by the clustered regularly interspaced short palindromic repeats interference system, inhibiting native fermentation pathways outcompeting this synthetic step. The present engineered strain exhibited a 15.7-fold increase in MCFA titer compared with that of the initial strain, and produced 15.67 g/L MCFAs from the biodiesel byproduct glycerol in 3-L bioreactor without exogenous feed of acetate or formate, representing the highest MCFA titer reported to date. This work demonstrates this artificial micro-aerobic metabolism has the potential to enable the cost-effective, large-scale production of fatty acids and other value-added reduced chemicals.

Cichoric Acid Prevents Free-Fatty-Acid-Induced Lipid Metabolism Disorders via Regulating Bmal1 in HepG2 Cells.

Cichoric acid (CA), a polyphenol component from Echinacea purpurea, exhibits preventive effects on liver lipid-metabolism disorders in obesity. This research aimed to determine the role of circadian rhythm signaling during the process of CA-attenuated lipid accumulation in hepatocytes. In the current study, CA treatments improved cell morphology changes and hepatic lipid levels, which were triggered by free fatty acids (2:1, oleate: palmitate) in a dose-dependent way. Besides, CA (200 µM) regulated the circadian rhythm expressions of clock genes and the relatively shallow daily oscillations. Moreover, silencing Bmal1 significantly blocked the p-Akt/Akt pathway to 80.1% ± 1.5% and the p-GSK3ß/GSK3ß pathway to 64.7% ± 2.8% ( p < 0.05). Furthermore, silencing Bmal1 elevated the expressions of FAS and ACC to 122.4% ± 5.6% and 114.9% ± 1.7% in protein levels ( p < 0.05) and to 166.5% ± 18.5% and 131.4% ± 5.5% in mRNA levels ( p < 0.05). Therefore, our results demonstrated that CA has a Bmal1 resistance to lipid accumulation by enhancing the Akt/GSK3ß signaling pathways and modulating the downstream expressions related to lipid metabolism, which indicated that CA might be useful as a natural and promising nonalcoholic fatty liver diseases (NAFLD) modulator.

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH.

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.

Engineering Escherichia coli to convert acetic acid to ß-caryophyllene.

BACKGROUND: Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce ß-caryophyllene by the engineered E. coli using acetic acid as the only carbon source. RESULTS: Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to ß-caryophyllene, acetyl-CoA synthase (ACS), ß-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance ß-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of ß-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and ß-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to ß-caryophyllene (gram to gram) reached 2.1%. The yield of ß-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield. CONCLUSIONS: In the present study, a novel biosynthetic pathway for ß-caryophyllene has been investigated by means of conversion of acetic acid to ß-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in ß-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for ß-caryophyllene synthesis.

Involvement of the TetR-Type Regulator PaaR in the Regulation of Pristinamycin I Biosynthesis through an Effect on Precursor Supply in Streptomyces pristinaespiralis.

UNLABELLED: Pristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two proteinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of streptogramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is understood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified as playing a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptional repressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator as PaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identical to the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given that phenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway of L-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription of paa genes in a ΔpaaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolic flux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI production of a ΔpaaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the ΔpaaR mutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis. IMPORTANCE: A better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomyces strain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes involved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) by affecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge, this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Considering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, it could be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli.

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C4 carboxylates into two molecules of acetyl-CoA without loss of CO2. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.

Acetate-dependent mechanisms of inborn tolerance to ethanol.

AIMS: To clarify the role of acetate in neurochemical mechanisms of the initial (inborn) tolerance to ethanol. METHODS: Rats with low and high inborn tolerance to hypnotic effect of ethanol were used. In the brain region homogenates (frontal and parietal cortex, hypothalamus, striatum, medulla oblongata) and brain cortex synaptosomes, the levels of acetate, acetyl-CoA, acetylcholine (AcH), the activity of pyruvate dehydrogenase (PDG) and acetyl-CoA synthetase were examined. RESULTS: It has been found that brain cortex of rats with high tolerance to hypnotic effect of ethanol have higher level of acetate and activity of acetyl-CoA synthetase, but lower level of acetyl-СCoA and activity of PDG. In brain cortex synaptosomes of tolerant rats, the pyruvate oxidation rate as well as the content of acetyl-CoA and AcH synthesis were lower when compared with intolerant animals. The addition of acetate into the medium significantly increased the AcH synthesis in synaptosomes of tolerant, but not of intolerant animals. Calcium ions stimulated the AcH release from synaptosomes twice as high in tolerant as in intolerant animals. Acetate eliminated the stimulating effect of calcium ions upon the release of AcH in synaptosomes of intolerant rats, but not in tolerant animals. As a result, the quantum release of AcH from synaptosomes in the presence of acetate was 6.5 times higher in tolerant when compared with intolerant rats. CONCLUSION: The brain cortex of rats with high inborn tolerance to hypnotic effect of ethanol can better utilize acetate for the acetyl-CoA and AcH synthesis, as well as being resistant to inhibitory effect of acetate to calcium-stimulated release of AcH. It indicates the metabolic and cholinergic mechanisms of the initial tolerance to ethanol.

Biochemical and genetic characterization of an auxotroph of Bacillus subtilis altered in the Acyl-CoA:acyl-carrier-protein transacylase.

We have analyzed a mutation of Bacillus subtilis (bfmB) that results in an acyl-CoA:acyl-carrier-protein transacylase with low affinity for branched acyl-CoA substrates; it maps in the acf-hisH region of the chromosome. The aceA mutation, present in the parent of the bfmB mutant, causes a deficiency in pyruvate dehydrogenase and maps in the pycA-pyrA region. Strains carrying the bfmB mutation synthesize branched-chain fatty acids at a rate sufficient for normal growth only if branched acyl-CoA precursors are present in the medium. They grow well if the medium is supplemented with 0.1 mM 2-methylbutyrate, isobutyrate or isovalerate, or with 1.0 mM isoleucine or valine; leucine does not support growth. Growth supported by valine and isoleucine is inhibited by butyrate and other straight short-chain fatty acids at concentrations (0.1 mM) which do not inhibit growth of the standard strain; the inhibition is prevented by short branched fatty acids which are converted to long-chain fatty acids appearing as activity of B. subtilis is controlled by separate enzymatic sites for the acyl-CoA precursors of branched and straight-chain fatty acids. Whether these sites are contained in one or two enzymes is not known.