Production Of Branched-chain Fatty Acids Research Articles

Microbial Fermentation of the Water-Soluble Fraction of Brewers’ Spent Grain for the Production of High-Value Fatty Acids

Brewers’ spent grain (BSG) constitutes the primary by-product of the brewing industry. The valorization of BSG from a circular economy perspective is of high industrial interest. The objective of this study was the exploitation of BSG for the microbial production of branched-chain fatty acids (BCFAs) and polyunsaturated fatty acids (PUFAs), representing two different classes of high-value fatty acids (FAs). In the present study, this waste material underwent treatment with hot water in an autoclave and the resultant extract was utilized for the preparation of a novel liquid medium (BSG medium) to be employed for microbial fermentation. Screening and subsequent scaling-up experiments confirmed the suitability of the BSG medium to support the microbial production of various high-value FAs. In particular, Streptomyces jeddahensis and Conidiobolus heterosporus could be employed for BCFAs production, Pythium ultimum and Mortierella alpina could be used to provide cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and arachidonic acid (ARA), whereas Mucor circinelloides, when grown in a BSG medium, was able to accumulate γ-linolenic acid (GLA).

Electrospray alginate microgels co-encapsulating degraded Konjac glucomannan and quercetin modulate human gut microbiota in vitro

Alginate microgels co-encapsulating degraded Konjac glucomannan (KGM60) underwent in vitro fecal fermentation and their effects on human microbiota and metabolites were investigated. KGM60 delayed quercetin release and enhanced phenolic metabolites production. Microgels co-encapsulating KGM60 and quercetin increased linear short chain fatty acid but decreased branched chain fatty acid production. Microgels encapsulated with quercetin with or without KGM60 decreased Firmicutes while increased Bacteroidetes over 24 h of fermentation, at genus level promoted Bacteroides growth at 24 h and decreased the abundance of Negativibacillus, Ruminococcus_NK4A214, and Christensenellaceae R_7. Faecalibacterium and Collinsella levels were exclusively promoted by microgels encapsulating KGM60 with or without quercetin, highlighting prebiotic effect of KGM60. Only microgels co-encapsulating both KGM60 and quercetin enhanced Dialister while inhibited Lachnoclostridium, indicating synergism between KGM60 and quercetin. Our study indicates that co-encapsulating KGM60 and quercetin in alginate microgel is effective in modulating human gut microbiota and metabolites production potentially beneficial to gut health.

Effect of Protein Fermentation Products on Gut Health Assessed in an In Vitro Model of Human Colon (TIM-2).

Western type of diets are characterized by high animal protein intake and are associated with various chronic inflammatory diseases. With a higher protein consumption, excess undigested protein will reach the colon and be subsequently metabolized by gut microbiota. Depending on the type of protein, fermentation in the colon generates different metabolites with varying biological effects. This study aims to compare the impact of protein fermentation products from different sources on gut health. Three high protein diets (vital wheat gluten [VWG], lentil, or casein) are submitted to the in vitro model of colon. Fermentation of excess lentil protein for 72 h results in highest production of short-chain fatty acids and lowest production of branched-chain fatty acids. Exposure of Caco-2 monolayers or Caco-2 monolayers co-cultured with THP-1 macrophages to luminal extracts of fermented lentil protein results in less cytotoxicity of Caco-2 monolayers and less damage to barrier integrity, when compared to VWG and casein. Lowest induction of interleukin-6 is observed in THP-1 macrophages after treatment with lentil luminal extracts, which is identified to be regulated by aryl hydrocarbon receptor signaling. The findings indicate that protein sources affect the health effects of high protein diet in the gut.

Hepatic and serum branched-chain fatty acid profile in patients with nonalcoholic fatty liver disease: A case-control study.

Alterations in the hepatic lipidome are a crucial factor involved in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate the serum and hepatic profile of branched-chain fatty acids (BCFAs) in patients with different stages of NAFLD. This was a case-control study performed in 27 patients without NAFLD, 49 patients with nonalcoholic fatty liver,and 17 patients with nonalcoholic steatohepatitis, defined by liver biopsies. Serum and hepatic levels of BCFAs were analyzed by gas chromatography-mass spectrometry. The hepatic expression of genes involved in the endogenous synthesis of BCFAs was analyzed byreal-time quantitative polymerase chain reaction (RT-qPCR). A significant increase in hepatic BCFAs was found in subjects with NAFLD compared with those without NAFLD; no differences were observed in serum BCFAs between study groups. Trimethyl BCFAs, iso-BCFAs, and anteiso-BCFAs were increased in subjects with NAFLD (either nonalcoholic fatty liver or nonalcoholic steatohepatitis) compared with those without NAFLD. Correlation analysis showed a relationship between hepatic BCFAs and the histopathological diagnosis of NAFLD, as well as other histological and biochemical parameters related to this disease. Gene expression analysis in liver showed that the mRNA levels of BCAT1, BCAT2, and BCKDHA were upregulated in patients with NAFLD. These results suggest that the increased production of liver BCFAs might be related to NAFLD development and progression.

Nutritional Value of Climate-Resilient Forage Species Sustaining Peri-Urban Dairy Cow Production in the Coastal Grasslands of Benin (West Africa).

Along the coast of West Africa, grazing ruminants rely on perennial forage species remaining in uncultivated plots, roadsides, and marshlands during the dry season. To assess the quality of these forages, thirteen drought-tolerant plants were harvested at the mature stage, and the samples were evaluated for chemical composition, in vitro fermentation characteristics, and metabolizable energy (ME) content. They are ten drought-tolerant grasses, including: Andropogon virginicus, Brachiaria deflexa, Cenchorus biflorus, Dactyloctenium aegyptium, Eragrostis tremula, Leptochloa caerulescens, Loudetia aroundinacea, Paspalum notatum, Paspalum vaginatum, Pennisetum purpureum, two perennial herbs, Chamaecrista rotundifolia, Zornia latifolia, and one multipurpose tree, Elaeis guineensis. Legume species had the highest nutritional value (highest crude protein and ME, and lowest neutral detergent fiber) of the species studied. In terms of the in vitro data, the gas produced after 120 h of incubation ranged from 149 mL/g in E. tremula to 185 mL/g in Paspalum. Z. latifoliaa and had the fastest rate of fermentation, producing half of the total gas in 19.5 h, whereas E. tremula required 49.9 h (p < 0.01). The production of branched-chain fatty acids (isobutyrate and isovalerate) was greatest for E. guineensis and the lowest in both Paspalum species (p < 0.01). The study suggests the need for the protein supplementation of the animals to ensure maximum forage utilization and to satisfy the nutrient requirements of ruminant livestock.

Feeding diversified protein sources exacerbates hepatic insulin resistance via increased gut microbial branched-chain fatty acids and mTORC1 signaling in obese mice

Animal models of human diseases are classically fed purified diets that contain casein as the unique protein source. We show that provision of a mixed protein source mirroring that found in the western diet exacerbates diet-induced obesity and insulin resistance by potentiating hepatic mTORC1/S6K1 signaling as compared to casein alone. These effects involve alterations in gut microbiota as shown by fecal microbiota transplantation studies. The detrimental impact of the mixed protein source is also linked with early changes in microbial production of branched-chain fatty acids (BCFA) and elevated plasma and hepatic acylcarnitines, indicative of aberrant mitochondrial fatty acid oxidation. We further show that the BCFA, isobutyric and isovaleric acid, increase glucose production and activate mTORC1/S6K1 in hepatocytes. Our findings demonstrate that alteration of dietary protein source exerts a rapid and robust impact on gut microbiota and BCFA with significant consequences for the development of obesity and insulin resistance.

Intestinal Health and Threonine Requirement of Growing Pigs Fed Diets Containing High Dietary Fibre and Fermentable Protein

Simple SummaryDietary components, such as fibre and protein, have significant impacts on nutrient requirements and intestinal health in pigs. The objectives of this study were to investigate the interactive effects of dietary fibre and fermentable protein on threonine requirement for protein deposition in growing pigs and to determine how these factors affect markers of intestinal health. In this study we used the nitrogen-balance approach to study the influence of high protein diets combined with high fibre on threonine requirement for protein deposition. We further used gene expression, fermentation metabolites (short and branched chain fatty acid concentration), and serum antioxidant status in these pigs as markers of intestinal health and function. We demonstrate that high fibre will indeed increase threonine requirement for protein deposition but can mitigate the negative effects of fermentable protein metabolites on intestinal health. These results will have implications for the development of dietary strategies to improve growth and overall health in pigs, including adjustments to dietary fibre, protein, and amino acid content that maximize pig growth, nutrient utilization, and intestinal health.Dietary fibre (DF) and fermentable crude protein (fCP) are dietary factors which affect nutrient utilization and intestinal health in pigs. A nitrogen (N)-balance study was conducted to determine the impact of DF and fCP on threonine (Thr) requirement for protein deposition (PD) and indicators of intestinal health. A total of 160 growing pigs (25 kg) were randomly assigned to 1 of 20 dietary treatments in a 2 × 2 × 5 factorial arrangement in a randomized complete block design with dietary fibre (low (LF) or high fibre (HF)], fCP [low (LfCP) or high fCP (HfCP)) and Thr (0.52, 0.60, 0.68, 0.76, or 0.82% standardized ileal digestible) as factors. Then, 4-day total urine and fecal collection was conducted, and pigs were euthanized for intestinal tissue and digesta sampling. Feeding high DF, regardless of fCP content, increased Thr requirement for PD (p < 0.05). High fCP, regardless of DF content, reduced Thr requirement for PD. Serum antioxidant capacity increased as dietary Thr level increased (p < 0.05). Cecal digesta short-chain fatty acids (SCFA) increased (p < 0.05) with HF and branched-chain fatty acids (BCFA) increased with HfCP and reduced with HF (p < 0.05). HfCP reduced (p < 0.05) mucin-2 (MUC2) expression in the colon of the HF but not the LF fed pigs and HF increased MUC2 in the LfCP but not the HfCP fed pigs. Feeding HF diet increased (p < 0.05) expression of zonula occludens-1 in the LfCP with no effect on HfCP fed pigs. Ammonia concentration in both cecum and colon increased (p < 0.05) in the HfCP fed pigs. Overall, high DF reduced the negative impact of HfCP on intestinal health, as indicated by alterations in SCFA and BCFA production and gut barrier gene expression. While increased dietary Thr content is required for PD in pigs fed high DF, feeding high fCP reduced Thr requirements.

Impact of Protein Intake in Older Adults with Sarcopenia and Obesity: A Gut Microbiota Perspective.

The continuous population increase of older adults with metabolic diseases may contribute to increased prevalence of sarcopenia and obesity and requires advocacy of optimal nutrition treatments to combat their deleterious outcomes. Sarcopenic obesity, characterized by age-induced skeletal-muscle atrophy and increased adiposity, may accelerate functional decline and increase the risk of disability and mortality. In this review, we explore the influence of dietary protein on the gut microbiome and its impact on sarcopenia and obesity. Given the associations between red meat proteins and altered gut microbiota, a combination of plant and animal-based proteins are deemed favorable for gut microbiota eubiosis and muscle-protein synthesis. Additionally, high-protein diets with elevated essential amino-acid concentrations, alongside increased dietary fiber intake, may promote gut microbiota eubiosis, given the metabolic effects derived from short-chain fatty-acid and branched-chain fatty-acid production. In conclusion, a greater abundance of specific gut bacteria associated with increased satiation, protein synthesis, and overall metabolic health may be driven by protein and fiber consumption. This could counteract the development of sarcopenia and obesity and, therefore, represent a novel approach for dietary recommendations based on the gut microbiota profile. However, more human trials utilizing advanced metabolomic techniques to investigate the microbiome and its relationship with macronutrient intake, especially protein, are warranted.

A prebiotic-enhanced lipid-based nutrient supplement (LNSp) increases Bifidobacterium relative abundance and enhances short-chain fatty acid production in simulated colonic microbiota from undernourished infants.

Undernutrition remains a public health problem in the developing world with an attributable under-five death proportion of 45%. Lower gut microbiota diversity and poor metabolic output are associated with undernutrition and new therapeutic paths may come from steering gut microbiota composition and functionality. Using a dynamic gut model, the Simulator of Human Intestinal Microbial Ecosystem (SHIME®), we investigated the effect of a lipid-based nutrient supplement enriched with prebiotics (LNSp), compared to LNS alone and control treatment, on the composition and metabolic functionality of fecal microbiota from three infants suffering from undernutrition. LNS elicited a significant increase in acetate and branched-chain fatty acid production, and a higher relative abundance of the genera Prevotella, Megasphaera, Acinetobacter, Acidaminococcus and Pseudomonas. In contrast, LNSp treatment resulted in a significant 9-fold increase in Bifidobacterium relative abundance and a decrease in that of potential pathogens and detrimental bacteria such as Enterobacteriaceae spp. and Bilophila sp. Moreover, the LNSp treatment resulted in a significantly higher production of acetate, butyrate and propionate, as compared to control and LNS. Our results suggest that provision of prebiotic-enhanced LNS to undernourished children could be a possible strategy to steer the microbiota toward a more beneficial composition and metabolic activity. Further in vivo investigations are needed to assess these effects and their repercussion on nutritional status.

Feruloylated oligosaccharides modulate the gut microbiota in vitro via the combined actions of oligosaccharides and ferulic acid

• Feruloylated oligosaccharides (FOs) have great prebiotic effects. • Ferulic acid has prebiotic effects which are different from oligosaccharides. • FOs showed prebiotic effects via the combined actions of oligosaccharides and ferulic acid. The effects of wheat bran feruloylated oligosaccharides (FOs) on the gut microbiota were studied. Furthermore, xylo-oligosaccharides (XOs) and ferulic acid (FA) as the two major moieties were used to demonstrate the structural influence of FOs on microbiota modulation effects. FOs promoted the phylum Firmicutes and Actinobacteria and decreased the relative abundance of Bacteroidetes and Proteobacteria. FA significantly increased the phylum Bacteroidetes. At the genus level, FOs promoted beneficial microorganism such as Bifidobacterium and Faecalibacterium , and inhibit the growth of disease-related bacteria such as Alistipes , Bilophila , Dorea , Enterobacteriaceae , and Oscillopira. FA significantly increased Bacteroides , Dialister and decreased Coprococcus , Enterobacteriaceae , Megamonas , Oscillospira . In terms of metabolites, FOs and XOs increased the content of short-chain fatty acids (SCFAs) but inhibited the production of branched-chain fatty acids (BCFAs). However, FA inhibited the production of both SCFAs and BCFAs. In conclusion, wheat bran FOs showed the prebiotic effects and further promoted the production of SCFAs due to the combined actions of oligosaccharides and FA moieties. Our findings highlighted that composition and structure should be taken into consideration for selecting FOs as prebiotics.

Effects of functional pasta ingredients on different gut microbiota as revealed by TIM-2 in vitro model of the proximal colon.

Diet-related modulation of gut microbiota and its metabolic activity represents an intriguing research context, particularly in the case of disorders related to imbalances in gut microbial communities. We here explored the effects of Bacillus coagulans GBI-30, 6086 (BC30), β-glucans, and innovative whole-grain pastas, with or without these functional ingredients, on gut microbiota from three groups of children, presenting different susceptibility to type 1 diabetes, by using the well-controlled TNO in vitro model of the proximal colon (TIM-2). Short- and branched-chain fatty acids production and microbiota composition were assessed by means of gas chromatography and 16S rRNA gene profiling, respectively. In most cases, in vitro dietary interventions caused microbiota-dependent modulations as a result of intergroup variability, but also specific changes in microbial groups were shared between the three microbiotas, highlighting specific diet-microbial taxa connections.

Impact of Deficiencies in Branched-Chain Fatty Acids and Staphyloxanthin in Staphylococcus aureus.

Staphylococcus aureus is a well-known human pathogen with the ability to cause mild superficial skin infections to serious deep-tissue infections, such as osteomyelitis, pneumonia, and infective endocarditis. A key to S. aureus infections and its pathogenicity is its ability to survive in adverse environments, especially at lower temperatures, by regulation of its cell membrane. Branched-chain fatty acids (BCFAs) and staphyloxanthin have been shown to regulate membrane fluidity and staphylococcal virulence. This study was conducted with the hypothesis that the simultaneous lack of BCFAs and staphyloxanthin will have a far greater implication on environmental survival and virulence of S. aureus. Lack of a functional branched-chain α-keto acid dehydrogenase (BKD) enzyme because of a mutation in the lpdA gene led to a decrease in the production of BCFAs, membrane fluidity, slower growth, and poor in vivo survival of S. aureus. A mutation in the crtM gene eliminated the production of staphyloxanthin but it did not affect membrane BCFA levels, fluidity, growth, or in vivo survival. A crtM:lpdA double mutant showed much slower growth and attenuation compared to individual mutants. The results of this study suggest that simultaneous targeting of the BCFA and staphyloxanthin biosynthetic pathways can be a strategy to control S. aureus infections.

Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls.

Colorectal cancer (CRC), a primary cause of morbidity and mortality worldwide is expected to rise in the coming years. A better understanding of the metabolic changes taking place during the disease progression is needed for effective improvements of screening strategies and treatments. In the present study, Nuclear Magnetic Resonance (NMR) metabolomics was used to quantify the absolute concentrations of metabolites in faecal extracts from two cohorts of CRC patients and healthy controls. The quantification of over 80 compounds revealed that patients with CRC had increased faecal concentrations of branched chain fatty acids (BCFA), isovalerate and isobutyrate plus valerate and phenylacetate but diminished concentrations of amino acids, sugars, methanol and bile acids (deoxycholate, lithodeoxycholate and cholate). These results suggest that alterations in microbial activity and composition could have triggered an increase in utilisation of host intestinal slough cells and mucins and led to an increase in BCFA, valerate and phenylacetate. Concurrently, a general reduction in the microbial metabolic function may have led to reduced levels of other components (amino acids, sugars and bile acids) normally produced under healthy conditions. This study provides a thorough listing of the most abundant compounds found in human faecal waters and presents a template for absolute quantification of metabolites. The production of BCFA and phenylacetate in colonic carcinogenesis warrants further investigations.

Human colonic microbiota modulation and branched chain fatty acids production affected by soy protein hydrolysate

SummarySeveral functional properties have been attributed to soy protein hydrolysates (SPHs); however, their gut fermentation needs to be investigated. This study aimed to determine the effect of hypo‐allergic pepsin‐educed SPH on the growth of gut microbiota, SCFA and BCFA production in a pH‐controlled, stirred, batch culture fermentation system compared with commercial SPH and soy protein isolate (SPI). The results showed that all substrates affected gut bacteria. SPH selectively increased the number of lactobacilli (log 9.61 to log 9.84) at 72‐h fermentation, but not for bifidobacteria. This is in accordance with increased BCFAs yield of 15.54 ± 0.00 mm (isobutyrate) and 465.59 ± 1.42 mm (isovalerate). The gut microbial balance index (GMBI) of SPH was 0.23, thus suggesting its gut microbial modulation. Therefore, SPH has potential for modulation or balance of gut microbiota, which may be used as functional ingredient for emerging market of nutraceuticals and nutri‐pharmaceutical industry.

Interrelationships between Fatty Acid Composition, Staphyloxanthin Content, Fluidity, and Carbon Flow in the Staphylococcus aureus Membrane.

Fatty acids play a major role in determining membrane biophysical properties. Staphylococcus aureus produces branched-chain fatty acids (BCFAs) and straight-chain saturated fatty acids (SCSFAs), and can directly incorporate exogenous SCSFAs and straight-chain unsaturated fatty acids (SCUFAs). Many S. aureus strains produce the triterpenoid pigment staphyloxanthin, and the balance of BCFAs, SCSFAs and staphyloxanthin determines membrane fluidity. Here, we investigated the relationship of fatty acid and carotenoid production in S. aureus using a pigmented strain (Pig1), its carotenoid-deficient mutant (Pig1ΔcrtM) and the naturally non-pigmented Staphylococcus argenteus that lacks carotenoid biosynthesis genes and is closely related to S. aureus. Fatty acid compositions in all strains were similar under a given culture condition indicating that staphyloxanthin does not influence fatty acid composition. Strain Pig1 had decreased membrane fluidity as measured by fluorescence anisotropy compared to the other strains under all conditions indicating that staphyloxanthin helps maintain membrane rigidity. We could find no evidence for correlation of expression of crtM and fatty acid biosynthesis genes. Supplementation of medium with glucose increased SCSFA production and decreased BCFA and staphyloxanthin production, whereas acetate-supplementation also decreased BCFAs but increased staphyloxanthin production. We believe that staphyloxanthin levels are influenced more through metabolic regulation than responding to fatty acids incorporated into the membrane.

Biological Functions of ilvC in Branched-Chain Fatty Acid Synthesis and Diffusible Signal Factor Family Production in Xanthomonas campestris.

In bacteria, the metabolism of branched-chain amino acids (BCAAs) is tightly associated with branched-chain fatty acids (BCFAs) synthetic pathways. Although previous studies have reported on BCFAs biosynthesis, more detailed associations between BCAAs metabolism and BCFAs biosynthesis remain to be addressed. In this study, we deleted the ilvC gene, which encodes ketol-acid reductoisomerase in the BCAAs synthetic pathway, from the Xanthomonas campestris pv. campestris (Xcc) genome. We characterized gene functions in BCFAs biosynthesis and production of the diffusible signal factor (DSF) family signals. Disruption of ilvC caused Xcc to become auxotrophic for valine and isoleucine, and lose the ability to synthesize BCFAs via carbohydrate metabolism. Furthermore, ilvC mutant reduced the ability to produce DSF-family signals, especially branched-chain DSF-family signals, which might be the main reason for Xcc reduction of pathogenesis toward host plants. In this report, we confirmed that BCFAs do not have major functions in acclimatizing Xcc cells to low temperatures.

Enzymatic Modification of Corn Starch Influences Human Fecal Fermentation Profiles.

Enzymatically modified starches have been widely used in food applications to develop new products, but information regarding digestion and fecal fermentation of these products is sparse. The objective of this study was to determine the fermentation properties of corn starch modified with α-amylase, amyloglucosidase, or cyclodextrin glycosyltransferase and the possible role of hydrolysis products. Samples differed in their digestibility and availability to be fermented by the microbiota, resulting in differences in microbial metabolites produced during in vitro fermentation. The presence or absence of hydrolysis products and gelatinization affected starch composition and subsequent metabolite production by the microbiota. Amyloglucosidase-treated starch led to the greatest production of short- and branched-chain fatty acid production by the microbiota. Results from this study could be taken into consideration to confirm the possible nutritional claims and potential health benefits of these starches as raw ingredients for food development.

Membrane fatty acid composition as a determinant of Listeria monocytogenes sensitivity to trans-cinnamaldehyde

trans-Cinnamaldehyde, the major compound of cinnamon essential oil, is a potentially interesting natural antimicrobial food preservative. Although a number of studies have addressed its mode of action, the factors that determine bacterial sensitivity or tolerance to trans-cinnamaldehyde are poorly understood. We report the detailed characterization of a Listeria monocytogenes Scott A trans-cinnamaldehyde hypersensitive mutant defective in IlvE, which catalyzes the reversible transamination of branched-chain amino acids to the corresponding short-chain α-ketoacids. This mutant showed an 8.4 fold extended lag phase during growth in sublethal concentrations (4 mM), and faster inactivation in lethal concentrations of trans-cinnamaldehyde (6 mM). trans-Cinnamaldehyde hypersensitivity could be corrected by genetic complementation with the ilvE gene and supplementation with branched-chain α-ketoacids. Whole-cell fatty acid analyses revealed an almost complete loss of anteiso branched-chain fatty acids (BCFAs), which was compensated by elevated levels of unbranched saturated fatty acids and iso-BCFAs. Sub-inhibitory concentrations of trans-cinnamaldehyde induced membrane fatty acid adaptations predicted to reduce membrane fluidity, possibly as a response to counteract the membrane fluidizing effect of trans-cinnamaldehyde. These results demonstrate the role of IlvE in BCFA production and the role of membrane composition as an important determinant of trans-cinnamaldehyde sensitivity in L. monocytogenes.

Engineering Escherichia coli to produce branched-chain fatty acids in high percentages

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.

Branched-chain alcohol formation by thermophilic bacteria within the genera of Thermoanaerobacter and Caldanaerobacter.

Fifty-six thermophilic strains including members of Caldanaerobacter, Caldicellulosiruptor, Caloramator, Clostridium, Thermoanaerobacter, and Thermoanaerobacterium, were investigated for branched-chain amino acid degradation in the presence of thiosulfate in batch culture. All of the Thermoanaerobacter and Caldanaerobacter strains (24) degraded the branched-chain amino acids (leucine, isoleucine, and valine) to a mixture of their corresponding branched-chain fatty acids and branched-chain alcohols. Only one Caloramator strain degraded the branched-chain amino acids to the corresponding branched-chain fatty acids. The ratio of branched-chain fatty acid production over branched-chain alcohol production for Thermoanaerobacter was 7.15, 6.61, and 11.53 for leucine, isoleucine, and valine, respectively. These values for Caldanaerobacter were 3.49, 4.13, and 7.31, respectively. This indicates that members within Caldanaerobacter produce proportionally more of the alcohols as compared with Thermoanaerobacter. No species within other genera investigated produced branched-chain alcohols from branched-chain amino acids in the presence of thiosulfate.