Production of (R)-mandelic acid from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations
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ABSTRACT:
SUBMITTER: Lukito B
PROVIDER: S-EPMC10992357 | biostudies-literature | 2021 Mar
REPOSITORIES: biostudies-literature
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Project description:Testosterone (TS) and its 1(2)-dehydrogenated derivative boldenone (BD) are widely used in medicine, veterinary science and as precursors in organic synthesis of many therapeutic steroids. Green production of these compounds is possible from androstenedione (AD) enzymatically, or from phytosterol (PS) using fermentation stages. In this study, the ascomycete Curvularia sp. VKM F-3040 was shown to convert androstadienedione (ADD, 4 and 10 g/L) to yield 97% and 78% (mol/mol) of BD, respectively. Based on its high 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, a novel cascade biotransformation of PS was developed for production of TS and BD. At the first stage, the strains of Mycolicibacterium neoaurum VKM Ac-1815D or M. neoaurum VKM Ac-1816D converted PS (5 or 10 g/L) into AD or ADD (each in a concentration of 2.5 or 5 g/L), respectively. At the second stage, mycelium of the fungus under the revealed optimal conditions reduced AD or ADD with more than 90% efficiency to form TS or BD, respectively. Based on transcriptome analysis, six candidate genes that might encode 17β-HSDs in the Curvularia sp. genome were revealed. Along with 17β-HSDs, the fungus possessed inducible P450cur 7-monooxygenase, which led to the accumulation of 7α-hydroxytestosterone (7α-OH-TS) as a major product from AD (up to 83% within 24 h after mycelium addition at the second stage of cascade biotransformation). The presence of protein synthesis inhibitor cycloheximide (CHX) prevented 7α/β-hydroxylation due to inhibition of de novo synthesis of the enzyme in the fungal cells. The results demonstrate the high biotechnological potential of the Curvularia sp. strain and open up prospects for the synthesis of valuable 17β-reduced and 7-hydroxylated steroids by cascade biotransformations.
| S-EPMC11677960 | biostudies-literature
Project description:Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheAfbr) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120 mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.
| S-EPMC6199770 | biostudies-literature
Project description:BACKGROUND: Mandelic acid (MA), an important component in pharmaceutical syntheses, is currently produced exclusively via petrochemical processes. Growing concerns over the environment and fossil energy costs have inspired a quest to develop alternative routes to MA using renewable resources. Herein we report the first direct route to optically pure MA from glucose via genetic modification of the L-phenylalanine pathway in E. coli. RESULTS: The introduction of hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis into E. coli led to a yield of 0.092 g/L S-MA. By combined deletion of competing pathways, further optimization of S-MA production was achieved, and the yield reached 0.74 g/L within 24 h. To produce R-MA, hydroxymandelate oxidase (Hmo) from Streptomyces coelicolor and D-mandelate dehydrogenase (DMD) from Rhodotorula graminis were co-expressed in an S-MA-producing strain, and the resulting strain was capable of producing 0.68 g/L R-MA. Finally, phenylpyruvate feeding experiments suggest that HmaS is a potential bottleneck to further improvement in yields. CONCLUSIONS: We have constructed E. coli strains that successfully accomplished the production of S- and R-MA directly from glucose. Our work provides the first example of the completely fermentative production of S- and R-MA from renewable feedstock.
| S-EPMC3182895 | biostudies-literature
Project description:Background(R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains.ResultsHerein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate.ConclusionsThe microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.
| S-EPMC9838050 | biostudies-literature
Project description:Ethylbenzene and styrene are air toxicants with widespread nonoccupational exposure sources, including tobacco smoke and diet. Ethylbenzene and styrene (EB/S) exposure was quantified from their common metabolites measured in spot urine samples obtained from participants (≥6 years old) in the 2005-2006 and 2011-2012 cycles of the National Health and Nutrition Examination Survey (NHANES; N = 4690). EB/S metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) were measured using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). MA and PGA were detected in 98.9% and 90.6% of tested urine specimens, respectively. Exclusive smokers had 2-fold and 1.6-fold higher median urinary MA and PGA, respectively, compared with non-users. Sampleweighted regression analysis among exclusive smokers showed that smoking 0.5 pack cigarettes per day significantly increased MA (+97.9 μg/L) and PGA (+69.3 μg/L), controlling for potential confounders. In comparison, exposure from the median daily dietary intake of grain products increased MA by 1.95 μg/L and was not associated with statistically significant changes in urinary PGA levels. Conversely, consuming vegetables and fruit was associated with decreased MA and PGA. These results confirm tobacco smoke as a major source of ethylbenzene and styrene exposure for the general U.S. population.
| S-EPMC6382531 | biostudies-literature
Project description:An activity-guided fractionation approach revealed several phenylpropanoid glycerol glucosides isolated from the bulbs of Lilium longiflorum Thunb. (Easter lily) with gluconeogenesis inhibitory activities. The strongest activity was observed for (2S)-1-O-p-coumaroyl-2-O-β-d-glucopyranosylglycerol (3), (2S)-1-O-caffeoyl-2-O-β-d-glucopyranosylglycerol (1), and (2R)-1-O-β-d-glucopyranosyl-2-O-p-coumaroylglycerol (2) with inhibitions of 51.2, 39.2, and 36.8%, respectively. The p-coumaroyl-based (3) and its acetylated derivative (5) exhibited differential inhibition activity (51.2% as compared to 3.6%), suggesting that natural acetylation decreases the hypoglycemic activity of these compounds. Direct structure-activity analysis of phenylpropanoid glycerol glucosides indicated that the hydroxylation pattern of the hydroxy cinnamic acid moiety and acetylation were responsible for the differences in activity. This is the first report of phenylpropanoid glycerol glucosides as a phytochemical class of hepatic glucose production inhibitors.
| S-EPMC6648405 | biostudies-literature
Project description:BackgroundPropionic acid fermentation from renewable feedstock suffers from low volumetric productivity and final product concentration, which limits the industrial feasibility of the microbial route. High cell density fermentation techniques overcome these limitations. Here, propionic acid (PA) production from glucose and a crude glycerol/glucose mixture was evaluated using Acidipropionibacterium acidipropionici, in high cell density (HCD) batch fermentations with cell recycle. The agro-industrial by-product, heat-treated potato juice, was used as N-source.ResultsUsing 40 g/L glucose for nine consecutive batches yielded an average of 18.76 ± 1.34 g/L of PA per batch (0.59 gPA/gGlu) at a maximum rate of 1.15 gPA/L.h, and a maximum biomass of 39.89 gCDW/L. Succinic acid (SA) and acetic acid (AA) were obtained as major by-products and the mass ratio of PA:SA:AA was 100:23:25. When a crude glycerol/glucose mixture (60 g/L:30 g/L) was used for 6 consecutive batches with cell recycle, an average of 35.36 ± 2.17 g/L of PA was obtained per batch (0.51 gPA/gC-source) at a maximum rate of 0.35 g/L.h, and reaching a maximum biomass concentration of 12.66 gCDW/L. The PA:SA:AA mass ratio was 100:29:3. Further addition of 0.75 mg/L biotin as a supplement to the culture medium enhanced the cell growth reaching 21.89 gCDW/L, and PA productivity to 0.48 g/L.h, but also doubled AA concentration.ConclusionThis is the highest reported productivity from glycerol/glucose co-fermentation where majority of the culture medium components comprised industrial by-products (crude glycerol and HTPJ). HCD batch fermentations with cell recycling are promising approaches towards industrialization of the bioprocess.
| S-EPMC10964606 | biostudies-literature
Project description:Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.
| S-EPMC9501478 | biostudies-literature
Project description:BackgroundThe microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol-1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield.ResultsBy changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol-1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol-1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase.ConclusionsThe data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.
| S-EPMC9148483 | biostudies-literature
Project description:Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.
| S-EPMC5428006 | biostudies-literature