Short Branched-chain Fatty Acids Research Articles

Transporter-Driven Engineering of a Genetic Biosensor for the Detection and Production of Short-Branched Chain Fatty Acids in Saccharomyces cerevisiae.

Biosensors can be used for real-time monitoring of metabolites and high-throughput screening of producer strains. Use of biosensors has facilitated strain engineering to efficiently produce value-added compounds. Following our recent work on the production of short branched-chain fatty acids (SBCFAs) in engineered Saccharomyces cerevisiae, here we harnessed a weak organic acid transporter Pdr12p, engineered a whole-cell biosensor to detect exogenous and intracellular SBCFAs and optimized the biosensor’s performance by varying PDR12 expression. We firstly constructed the biosensor and evaluated its response to a range of short-chain carboxylic acids. Next, we optimized its sensitivity and operational range by deletion and overexpression of PDR12. We found that the biosensor responded to exogenous SBCFAs including isovaleric acid, isobutyric acid and 2-methylbutanoic acid. PDR12 deletion enhanced the biosensor’s sensitivity to isovaleric acid at a low concentration and PDR12 overexpression shifted the operational range towards a higher concentration. Lastly, the deletion of PDR12 improved the biosensor’s sensitivity to the SBCFAs produced in our previously engineered SBCFA-overproducing strain. To our knowledge, our work represents the first study on employing an ATP-binding-cassette transporter to engineer a transcription-factor-based genetic biosensor for sensing SBCFAs in S. cerevisiae. Our findings provide useful insights into SBCFA detection by a genetic biosensor that will facilitate the screening of SBCFA-overproducing strains.

Research Progress on the Undesirable Flavor in Natto

Cardiovascular disease is the chronic disease with the most deaths in our country. And natto products, made from soybean by fermented by Bacillus subtilis, have high-efficiency thrombolytic, lipid-lowering, and blood pressure-lowering effects. However, the inherent undesirable flavor of natto restricts its development in the Chinese food industry. This article mainly reviews the flavor improvement methods of natto in recent years, in theaspects of strain, technology and seasoning, and summarizes the undesirable flavor substances in natto, involving in free ammonia, pyrazine and branched-chain short fatty acids. This review aims to provide scientific basis for promoting the development of natto with good quality and flavor.

Branched chain fatty acids in the flavour of sheep and goat milk and meat: A review

• The meat and milk from sheep and goats possess unique, variable and complex flavour profiles. • The different flavour profiles result from complex interactions of genetics, age, sex, and nutrition. • Significant gaps exist in our understanding of those relationships. • Analytical techniques are being used to understand the flavour of goat and sheep meat, and milk. Globally, around 1.5 billion sheep and goats provide meat and milk on an annual basis and are important sources of nutrition as well as economic subsistence. The popularity of sheep and goat meat, along with milk, and related products occurs due to their unique nutritional properties, physicochemical composition, and sensory attributes while having species-specific flavours compared to other red meats and dairy products. The overall volatile flavour profile and sensory characteristics of the products derived from sheep and goats are key differentiators in the market. However, the flavour variation can lead to inconsistent consumer experiences. A complex relationship exists between factors such as breed, sex, and age along with nutrition which contribute to the overall flavour. Additionally, regional expectations of flavour can also influence consumers. The characteristic flavour associated with dairy products derived from sheep and goat, has a tremendous impact on the overall product quality and acceptability, distinct from that found with cow milk. This review focuses on the short branched chain fatty acids identified as relevant to the flavour associated with milk and meat from sheep and goats, the synthesis of these compounds as well as the pre- and post-gate factors which affect their accumulation in meat and milk. It will also present the pros and cons of classical and emerging analytical techniques used for their determination.

High-level production of short branched-chain fatty acids from waste materials by genetically modified Bacillus licheniformis

Short branched-chain fatty acids (SBCFAs) are multi-functional platform chemicals used in many fields. Currently, SBCFAs are produced mainly by chemical synthesis, which is high cost and lead to environmental pollution. The aim of this study was to achieve high-level production of SBCFAs from waste materials, bean dreg and crude glycerol. The Bacillus licheniformis DWc9n∗ was genetically modified by overexpression of SBCFAs synthesis genes via replacement of native promoter of bkd operon, the mutant strain DWc9n∗-PbacA produced 4.68 g/L of SBCFAs, increasing by 1.98-fold compared to wild-type strain. SBCFAs concentration was further increased to 7.85 g/L through process optimization. In a 5-L batch fermenter, the mutant showed SBCFAs production with high concentration (8.37 g/L) and productivity (0.20 g/L/h), which is the highest level of SBCFAs production based on low-value substrates fermentation. This is the first study describing efficient SBCFAs production by the modified B. licheniformis strain from bean dreg and crude glycerol.

Identification of a Key Gene Involved in Branched-Chain Short Fatty Acids Formation in Natto by Transcriptional Analysis and Enzymatic Characterization in Bacillus subtilis.

Natto as a fermented soybean product has many health benefits for human due to its rich nutritional and functional components. However, the unpleasant odor of natto, caused by the formation of branched-chain short fatty acids (BCFAs), prohibits the wide acceptance of natto products. This work is to identify the key gene of BCFAs formation and develop the guidance to reduce natto odor. Transcriptional analysis of BCFAs synthesis pathway genes was conducted in two Bacillus subtilis strains with obvious different BCFAs synthesis abilities. The transcriptional levels of bcd, bkdAA, and ptb in B. subtilis H-9 were 2.7-fold, 0.7-fold, and 8.9-fold higher than that of B. subtilis H-4, respectively. Therefore, the ptb gene with the highest transcriptional change was considered as the key gene in BCFAs synthesis. The ptb encoded enzyme Ptb was further characterized by inducible expression in Escherichia coli. The recombinant Ptb protein (about 32 kDa) was verified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. The catalysis functions of Ptb were confirmed on substrates of isovaleryl-CoA and isobutyryl-CoA, and the higher catalysis efficiency of Ptb on isovaleryl-CoA explained the higher level of isovaleric acid in natto. The optimal activities of Ptb were observed at 50 °C and pH 8.0, and the enzymatic activity was inhibited by Ca2+, Zn2+, Ba2+, Mn2+, Cu2+, SDS, and EDTA. Collectively, this study reports a key gene responsible for BCFAs formation in natto fermentation and provides potential strategies to solve the odor problem.

Decreased formation of branched-chain short fatty acids in Bacillus amyloliquefaciens by metabolic engineering

To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared. By knockout of the ptb gene, 1.01g BCFAs kg-1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27g BCFAskg-1). Moreover, no significant difference was found in the DNJ concentration (0.7gkg-1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12. HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

Lactobacillus paracasei comparative genomics: towards species pan-genome definition and exploitation of diversity.

Lactobacillus paracasei is a member of the normal human and animal gut microbiota and is used extensively in the food industry in starter cultures for dairy products or as probiotics. With the development of low-cost, high-throughput sequencing techniques it has become feasible to sequence many different strains of one species and to determine its “pan-genome”. We have sequenced the genomes of 34 different L. paracasei strains, and performed a comparative genomics analysis. We analysed genome synteny and content, focussing on the pan-genome, core genome and variable genome. Each genome was shown to contain around 2800–3100 protein-coding genes, and comparative analysis identified over 4200 ortholog groups that comprise the pan-genome of this species, of which about 1800 ortholog groups make up the conserved core. Several factors previously associated with host-microbe interactions such as pili, cell-envelope proteinase, hydrolases p40 and p75 or the capacity to produce short branched-chain fatty acids (bkd operon) are part of the L. paracasei core genome present in all analysed strains. The variome consists mainly of hypothetical proteins, phages, plasmids, transposon/conjugative elements, and known functions such as sugar metabolism, cell-surface proteins, transporters, CRISPR-associated proteins, and EPS biosynthesis proteins. An enormous variety and variability of sugar utilization gene cassettes were identified, with each strain harbouring between 25–53 cassettes, reflecting the high adaptability of L. paracasei to different niches. A phylogenomic tree was constructed based on total genome contents, and together with an analysis of horizontal gene transfer events we conclude that evolution of these L. paracasei strains is complex and not always related to niche adaptation. The results of this genome content comparison was used, together with high-throughput growth experiments on various carbohydrates, to perform gene-trait matching analysis, in order to link the distribution pattern of a specific phenotype to the presence/absence of specific sets of genes.

Faecal Microbiota and Short-Chain Fatty Acid Levels in Faeces from Infants with Cow‘s Milk Protein Allergy

Background: The present study was designed to compare the faecal microbiota and concentrations of faecal short-chain fatty acid and ammonia between healthy and cow’s milk protein allergic (CMPA) infants. Methods: The population comprised 92 infants aged 2–12 months who were nonallergic (n = 46) or diagnosed as having CMPA (n = 46). Faecal samples were analyzed by fluorescent in situ hybridization and flow cytometry, using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. Acetic, propionic, butyric, isocaproic and branched-chain short fatty acids (BCSFA) were measured by gas-liquid chromatography, lactate by enzymatic reaction, and pH and ammonia levels were determined. Results: CMPA infant faeces had significantly higher proportions of the Clostridium coccoides group and Atopobium cluster and a higher sum of the proportions of the different bacterial groups in comparison to healthy infant faeces. Faecal pH and ammonia did not significantly differ between CMPA and healthy infants. Faeces concentrations and percentages of butyric acid and BCSFA were higher in CMPA infants than in healthy infants. Conclusions: The findings clearly set a link between a dysbiosis in gut microbiota composition and the pathogenesis of CMPA. No single species or genus appeared to play an essential role, but dysbiosis led to biomarkers of CMPA among bacterial fermentation products.

Production of branched-chain aroma compounds byPropionibacterium freudenreichii: links with the biosynthesis of membrane fatty acids

Short branched-chain fatty acids (BCFAs) are cheese flavour compounds, which result from the conversion of branched-chain amino acids (BCAAs). In Swiss cheese, the production of short BCFAs is mainly performed by Propionibacterium freudenreichii and is strain dependent. Our aim was to investigate the possible links between the biosynthesis of short BCFAs and membrane BCFAs in P. freudenreichii. Short and membrane BCFAs were analysed by gas chromatography-mass spectrometry. Two strains differing in their capacities to release short BCFAs were selected. Tri-deuterated-labelled leucine was used in both strains as a precursor of short extracellular iso-BCFAs and of membrane iso-BCFAs. The proportions of anteiso : iso BCFAs synthesized varied as function of the BCAAs provided in the growth medium, from 72 : 28 to 100 : 0, with leucine and valine, and with isoleucine as sole BC precursors, respectively. The branching pattern of short BCFAs exactly matched that of membrane BCFAs, whatever the exogenous BCAAs provided. The biosynthesis of short BCFAs is closely related to that of membrane BCFAs in P. freudenreichii. The biosynthesis of short BCFAs in P. freudenreichii depends more on the strain than on the presence of exogenous BC precursors.

Factors affecting the concentration of short branched-chain fatty acids in sheep fat

The concentration of short branched-chain fatty acids, specifically 4-methyloctanoic acid (MOA) and 4-methylnonanoic acid (MNA), in subcutaneous fat were determined in five genotypes (Poll Dorset × Border Leicester Merino, Poll Dorset × Merino, Poll Dorsetmuscling × Merino, Merino × Merino, Border Leicester × Merino) of Australian sheep. In total, 264 ewes and wethers, either 8 or 22 months of age, were selected for genotype, age and gender. These animals were sired by 16 rams that were selected for growth based on estimated breeding values (EBVs), except for one group (Poll Dorsetmuscling × Merino), which was selected for muscling based on EBVs. There was a strong relationship between the concentration of MOA and MNA at 8 and 22 months of age. There were no differences between lambs sired by Poll Dorset rams selected for muscling or growth. The 8- and 22-month-old Poll Dorset × Merino sheep had higher levels of MOA and MNA compared with the other genotypes (P < 0.001). At 8 months of age, the wethers had higher levels of MOA and MNA (P < 0.001) than the ewes of the same genotype. There was a strong relationship (P < 0.001) between MOA and MNA and the fat and lean percentage for 22-month-old ewes. Effects of sire, dam and slaughter day (at the same slaughter age) on MOA and MNA were also found. The results suggest that there is good feasibility for selection against animals at risk of possessing higher levels of short branched-chain fatty acids.

The Peroxisomal Acyl-CoA Thioesterase Pte1p from Saccharomyces cerevisiae Is Required for Efficient Degradation of Short Straight Chain and Branched Chain Fatty Acids

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Evolution of the biosynthesis of the branched-chain amino acids.

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

The esg locus of Myxococcus xanthus encodes the E1α and E1β subunits of a branched‐chain keto acid dehydrogenase

The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development. DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1 alpha and E1 beta subunits) of a branched-chain keto acid dehydrogenase (BCKAD) that is involved in branched-chain amino acid (BCAA) metabolism. The properties of an esg::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild-type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched-chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched-chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg-encoded enzyme is involved in the synthesis of branched-chain fatty acids during vegetative growth, and these compounds are used later in cell-cell signalling during development.

Properties of Bacillus acidocaldarius mutants deficient in ?-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in ω-cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing ω-cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.

Fatty Acid-requiring Mutant of Bacillus subtilis Defective in Branched Chain α-Keto Acid Dehydrogenase

A new mutant of Bacillus subtilis is described lacking the activity of branched chain α-keto acid dehydrogenase. The mutant requires short branched chain fatty acids biosynthetically related to leucine, isoleucine, and valine for growth. Long branched chain fatty acids derived from these short molecules can also serve as growth factors. One type of branched chain fatty acid, however, is sufficient to support growth of the mutant at 37°, although wild type cells of B. subtilis normally contain three different pairs of long branched chain fatty acids. Therefore, this mutant allows extensive alterations of the fatty acid composition in membranes of B. subtilis. Straight chain fatty acids do not support growth. Out of a series of tested analogues, 2-methylvalerate, 2-ethyl butyrate, and trimethylacetate were found to be active in vivo as primer molecules of branched chain fatty acid biosynthesis. The chain-lengthened products derived from these primer substrates were characterized by mass spectrometry.