D-phenyllactic Acid Research Articles

Innovative strategy of ZIF-90 for co-immobilization of whole cells and enzymes in biocatalytic D-phenyllactic acid synthesis.

In this study, we endeavored to catalyze the biosynthesis of D-phenyllactic (D-PLA) from L-Phenylalanine (L-Phe) through a one-pot method. However, the crucial enzymes for the biosynthesis of phenylpyruvate (PPA), amino acid oxidase (L-AAD), is a membrane-bound protein. Herein, we proposed a novel co-immobilization strategy of whole cells and enzymes, integrating them into ZIF-90 to achieve efficient biosynthesis of D-PLA. Consequently, we embarked on integrating both enzyme and E. coli into ZIF-90, ultimately obtaining the novel biocatalyst E. coli/LDH@ZIF-90. This achievement facilitated the cascade reaction between LDH and E. coli, enabling a streamlined one-pot bioconversion process. The morphology and structure of E. coli/LDH@ZIF-90 were thoroughly characterized using a range of methods, including XRD, SEM, FT-IR, CLSM, and XPS, which confirmed that the material had been successfully synthesized. Further activity experiments revealed that E. coli/LDH@ZIF-90 exhibited good stability even under harsh conditions. Additionally, the biocatalyst retained 76% of its initial catalytic activity after completing six cycles. Moreover, when utilized for the biosynthesis of D-PLA, this system demonstrated an impressive conversion rate of 85.2% after 12h. The successful cascade catalysis from L-Phe to D-PLA underscored the potential of the enzyme-cell cascade catalytic system, offering valuable insights for its potential industrial applications.

Corrigendum: SERS-based microdroplet platform for high-throughput screening of Escherichia coli strains for the efficient biosynthesis of D-phenyllactic acid

Corrigendum: SERS-based microdroplet platform for high-throughput screening of Escherichia coli strains for the efficient biosynthesis of D-phenyllactic acid

Cascade synthesis of D-phenyllactic acid using amorphous metal-organic framework-modified Escherichia coli

D-phenyllactic acid is an important medical intermediate and a potential green preservative. Due to its extensive use and high demand, there has been significant research interest in developing efficient synthesis methods. In this study, we aimed to efficiently produce D-phenyllactic acid from L-phenylalanine through a two-step cascade reaction using a coupled catalytic system consisting of two bacteria strains (E. coli/pET28a-lrldh and E. coli/pET28a-ladd2). To enhance the stability of this catalytic system, we encapsulated the two recombinant E. coli strains with amorphous ZIF-90, which was thoroughly characterized using various methods including SEM, FT-IR, TGA, XRD, XPS, and CLSM analysis. Compared to free cells, the immobilized living materials (amorphous ZIF-90@E. coli) showed good stability in harsh environments and superior recyclability. When applied to the production of D-PLA, the amorphous ZIF-90@E. coli system achieved a yield of 9.00 g/L and a conversion of 89.4 %. This successful coupling catalysis from L-phenylalanine to D-PLA indicates the potential of aZIF-90@E. coli for biosynthesis of D-phenyllactic acid in future industrial applications.

Enhanced catalytic activity and stability of lactate dehydrogenase for cascade catalysis of D-PLA by rational design

Serving as a vital medical intermediate and an environmentally-friendly preservative, D-PLA exhibits substantial potential across various industries. In this report, the urgent need for efficient production motivated us to achieve the rational design of lactate dehydrogenase and enhance catalytic efficiency. Surprisingly, the enzymatic properties revealed that a mutant enzyme, LrLDHT247I/D249A/F306W/A214Y (LrLDH-M1), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of Kcat. Correspondingly, molecular docking analysis provided a supporting explanation for the lower Km and higher Kcat/Km of the mutant enzyme. Thermostability analysis exhibited increased half-lives and the deactivation rate constants decreased at different temperatures (1.47–2.26-fold). In addition, the mutant showed excellent resistance abilities in harsh environments, particularly under acidic conditions. Then, a two-bacterium (E. coli/pET28a-lrldh-M1 and E. coli/pET28a-ladd) coupled catalytic system was developed and realized a significant conversion rate (77.7%) of D-phenyllactic acid, using 10 g/L L-phenylalanine as the substrate in a two-step cascade reaction.

SERS-based microdroplet platform for high-throughput screening of Escherichia coli strains for the efficient biosynthesis of D-phenyllactic acid.

D-Phenyllactic acid (D-PLA) is a potent antimicrobial typically synthesized through chemical methods. However, due to the complexity and large pollution of these reactions, a simpler and more eco-friendly approach was needed. In this study, a strain for D-PLA biosynthesis was constructed, but the efficiency was restricted by the activity of D-lactate dehydrogenase (DLDH). To address this issue, a DLDH mutant library was constructed and the Surface-Enhanced Raman Spectroscopy (SERS) was employed for the precise quantification of D-PLA at the single-cell level. The TB24 mutant exhibited a significant improvement in D-PLA productivity and a 23.03-fold increase in enzymatic activity, which was attributed to the enhanced hydrogen bonding and increased hydrophobicity within the substrate-binding pocket. By implementing multi-level optimization strategies, including the co-expression of glycerol dehydrogenase (GlyDH) with DLDH, chassis cell replacement, and RBS engineering, a significant increase in D-PLA yields was achieved, reaching 128.4g/L. This study underscores the effectiveness of SERS-based microdroplet high-throughput screening (HTS) in identifying superior mutant enzymes and offers a strategy for large-scale D-PLA biotransformation.

Engineered Escherichia coli Consortia Function in a Programmable Pattern for Multiple Enzymatic Biosynthesis.

Coordinating microbial consortia to realize complex synthetic pathways is an area of great interest in the rapidly growing field of biomanufacturing. This work presents a programmable method for assembling living cells based on the surface display of affinity groups, enabling whole-cell catalysis with optimized catalytic efficiency through the rational arrangement of cell assemblies and enzymes. In the context of d-phenyllactic acid (d-PLA) synthesis, four enzymes were rationally arranged considering substrate channeling and protein expression levels. The production efficiencies of d-PLA catalyzed by engineered microbial consortia were 1.31- and 2.55-fold higher than those of biofilm and whole-cell catalysts, respectively. Notably, substrate channeling was identified between the coimmobilized rate-limiting enzymes, resulting in a 3.67-fold improvement in catalytic efficiency compared with hybrid catalysts (free enzymes coupled with whole-cell catalysts). The highest yield of d-PLA catalyzed by microbial consortia was 102.85 ± 3.39 mM with 140 mM benzaldehyde as the substrate. This study proposes a novel approach to cell enzyme assembly for coordinating microbial consortia in multiple enzymatic biosynthesis processes.

One-pot encapsulation of lactate dehydrogenase and Fe3O4 nanoparticles into a metal-organic framework: A novel magnetic recyclable biocatalyst for the synthesis of D-phenyllactic acid.

The main challenges in bio-catalysis of d-phenyllactic acid (D-PLA) are poor tolerance of lactate dehydrogenase (LDH) to harsh environmental conditions and inability to recycle the catalyst. A novel magnetic framework composite was prepared as solid support for the immobilization of enzymes via one-pot encapsulation in this study. LDH/MNPs@MAF-7 was synthesized by the one-pot encapsulation of both LDH and magnetic nanoparticles (MNPs) in MAF-7. The LDH/MNPs@MAF-7 showed stable biological activity for the efficient biosynthesis of D-PLA. The structure and morphology of LDH/MNPs@MAF-7 were systematically characterized by SEM, FT-IR, XRD, VSM, XPS, TGA and N2 sorption. These indicated that LDH/MNPs@MAF-7 was successfully synthesized, exhibiting enhanced resistance to acid and alkali, temperature and organic solvents. Furthermore, the bio-catalyst could be separated easily using a magnet, and the reusability was once considerably expanded with 80% of enzyme activity last after eight rounds of recycling. Therefore, LDH/MNPs@MAF-7 could be used as a potential biocatalyst for the biosynthesis of D-PLA due to its good stability and recovery properties.

A two-enzyme system in an amorphous metal-organic framework for the synthesis of D-phenyllactic acid.

In this study, we synthesized an amorphous metal-organic framework by adjusting the concentration of precursors, and established a two-enzyme system consisting of lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH), which successfully achieved coenzyme recycling, and applied it to the synthesis of D-phenyllactic acid (D-PLA). The prepared two-enzyme-MOF hybrid material was characterized using XRD, SEM/EDS, XPS, FT-IR, TGA, CLSM, etc. In addition, reaction kinetic studies indicated that the MOF-encapsulated two-enzyme system exhibited faster initial reaction velocities than free enzymes due to its amorphous ZIF-generated mesoporous structure. Furthermore, the pH stability and temperature stability of the biocatalyst were evaluated, and the results indicated a significant improvement compared to the free enzymes. Moreover, the amorphous structure of the mesopores still maintained the shielding effect and protected the enzyme structure from damage by proteinase K and organic solvents. Finally, the remaining activity of the biocatalyst for the synthesis of D-PLA reached 77% after 6 cycles of use, and the coenzyme regeneration still maintained at 63%, while the biocatalyst also retained 70% and 68% residual activity for the synthesis of D-PLA after 12 days of storage at 4 °C and 25 °C, respectively. This study provides a reference for the design of MOF-based multi-enzyme biocatalysts.

Untargeted metabolomics revealed the key metabolites in milk fermented with starter cultures containing Lactobacillus plantarum CCFM8610

This study employed untargeted metabolomics to reveal the metabolic changes in fermented milk during storage. According to the sparse partial least squares-discriminant analysis (sPLS-DA), Lactobacillus plantarum CCFM8610 was confirmed to be critical for the metabolic profile of fermented milk. Five of the forty-three metabolites, namely benzoic acid, 6-hydroxycaproic acid, d-phenyllactic acid, hippuric acid, and N-oleoylethanolamine, were identified as the key metabolites related to L. plantarum CCFM8610. This study illustrated that L. plantarum CCFM8610, as a probiotic and co-cultivated starter culture, was critical for the functional properties and preservative qualities of fermented milk during storage. Overall, these metabolites had potential impact on the storage properties and physiological functions of fermented milk, which provides a valuable reference for improving the quality of fermented milk products.

Lactate dehydrogenase encapsulated in a metal-organic framework: A novel stable and reusable biocatalyst for the synthesis of D-phenyllactic acid

In this study, we synthesized a novel biocatalyst by encapsulating lactate dehydrogenase (LDH) in the metal-organic framework ZIF-90 by one-pot embedding. It showed strong biological activity for efficient synthesis of D-phenyllactic acid (D-PLA). The morphology and structure of LDH@ZIF-90 was systematically characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, confocal laser scanning microscopy (CLSM) and gas sorption. According to thermogravimetric analysis (TGA), the enzyme loading of the biocatalyst was 3 %. The Michaelis–Menten constant (Km) and maximal reaction rate (Vmax) of LDH@ZIF-90 were similar to those of free LDH, which proved that ZIF-90 had good biocompatibility to encapsulate LDH. At the same time, LDH@ZIF-90 exhibited enhanced tolerance to temperature, pH and organic solvents, and its reusability was greatly improved with 68 % of initial enzyme activity remaining after 7 rounds of recylcing. Overall, LDH encapsulated in ZIF-90 may be an economically competitive and environmentally friendly novel biocatalyst for the synthesis of D-PLA.

UPLC-Q-TOF-MS based metabolomics and chemometric analyses for green tea fermented with Saccharomyces boulardii CNCM I-745 and Lactiplantibacillus plantarum 299V

UPLC-Q-TOF-MS was employed to analyse the non-volatile components of green teas fermented with probiotic yeast (Saccharomyces boulardii) and lactic acid bacteria (LAB, Lactiplantibacillus plantarum). The flavone glycosides in yeast-fermented and stored tea decreased significantly, together with the increases of flavone aglycones and other simple flavone glycosides. LAB-fermented tea presented different flavone glycoside profiles; in which, both C-glycosides and O-glycosides decreased and the flavone aglycones were further degraded. The profiles of flavone glycosides and aglycones in co-cultured tea differed from that in yeast- or LAB-fermented tea; less glycosides were degraded but a greater number of aglycones were produced. Two unique LAB metabolites with bioactive and antifungal properties, D-phenyllactic acid (PLA) and p-OH-PLA, were found in both L. plantarum and co-cultured teas, and the co-fermentation showed a synergic effect on the production of these two compounds that would enhance the quality and preservation of fermented teas.

Using Unnatural Protein Fusions to Engineer a Coenzyme Self-Sufficiency System for D-Phenyllactic Acid Biosynthesis in Escherichia coli

The biosynthetic production of D-penyllactic acid (D-PLA) is often affected by insufficient supply and regeneration of cofactors, leading to high production cost, and difficulty in industrialization. In this study, a D-lactate dehydrogenase (D-LDH) and glycerol dehydrogenase (GlyDH) co-expression system was constructed to achieve coenzyme NADH self-sufficiency and sustainable production of D-PLA. Using glycerol and sodium phenylpyruvate (PPA) as co-substrate, the E. coli BL21 (DE3) harboring a plasmid to co-express LfD-LDH and BmGlyDH produced 3.95 g/L D-PLA with a yield of 0.78 g/g PPA, similar to previous studies. Then, flexible linkers were used to construct fusion proteins composing of D-LDH and GlyDH. Under the optimal conditions, 5.87 g/L D-PLA was produced by expressing LfD-LDH-l3-BmGlyDH with a yield of 0.97 g/g PPA, which was 59.3% increased compared to expression of LfD-LDH. In a scaled-up reaction, a productivity of 5.83 g/L/h was reached. In this study, improving the bio-catalytic efficiency by artificial redox self-equilibrium system with a bifunctional fusion protein could reduce the bio-production cost of D-PLA, making this bio-production of D-PLA a more promising industrial technology.

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39kDa. It is active at 40°C and pH 6.5 and stable in a neutral to alkaline environment below 35°C. The kinetic constants, including maximal reaction rate (V max), apparent Michaelis-Menten constant (K m), turnover number (K cat) and catalytic efficiency (K cat/K m) for phenylpyruvic acid were 1.95 U mg-1, 2.83mM, 12.29s-1, and 4.34mM-1s-1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5gl-1 d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2gl-1 d-1. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals.

Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae.

d-Phenyllactic acid is a versatile natural organic acid, which is used as an antiseptic agent, monomer of the biodegradable material poly-phenyllactic acid and in the synthesis chiral intermediate of pharmaceuticals. In this report, the novel NADH-dependent d-lactate dehydrogenase LrLDH was identified by screening a shotgun genome of Lactobacillus rossiae. To improve cofactor regeneration, the Exiguobacterium sibiricum glucose dehydrogenase EsGDH was overexpressed together with LrLDH in E. coli BL21(DE3)-pCDFDuet-1-gdh-ldh. The total enzyme activity in the fermentation broth of E. coli BL 21(DE3)-pCDFDuet-1-gdh-ldh peaked at 2359.0Ul-1 when induced by 10gl-1 lactose at 28°C and 150rpm for 14h. The biocatalytic reduction of sodium phenylpyruvate to d-phenyllactic acid was successfully carried out using whole cells of the engineered E. coli. Under the optimized biocatalysis conditions, 50gl-1 sodium phenylpyruvate was completely converted to d-phenyllactic acid with a space-time yield and enantiomeric excess of 262.8gl-1day-1 and > 99.5%, respectively. To our best knowledge, it is the highest productivity reported to date, with great potential for the mass production of d-phenyllactic acid.

Health benefit of fermented food uncovered

Fermented foods are known to be good for you, and good old-fashioned sauerkraut made by fermenting cabbage with lactic acid bacteria is no exception. Claudia Stäubert and colleagues at Leipzig University were investigating hydroxycarboxylic acid (HCA) receptors when they discovered that a molecule produced by sauerkraut bacteria, d-phenyllactic acid, binds strongly to one of those receptors, HCA3 (PLOS Genet. 2019, DOI: 10.1371/journal.pgen.1008145). The team fed volunteers sauerkraut and found high levels of d-phenyllactic acid in their blood and urine, high enough to activate the volunteers’ immune cells. This observation suggests that sauerkraut plays a physiological role as well as a nutritional one, a finding that Thue Schwartz of the University of Copenhagen says is “truly interesting.” HCA3 is found in only humans and apes. By using bioinformatics, Stäubert’s team showed that the gene for HCA3 entered the ape genome around the same time as did the gene for alcohol dehydrogenase,

Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.

Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol

The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A “bridge” between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals.

Enzymatic preparation of d-phenyllactic acid at high space-time yield with a novel phenylpyruvate reductase identified from Lactobacillus sp. CGMCC 9967

An NADH-dependent phenylpyruvate reductase (LaPPR) was identified through screening the shotgun library of Lactobacillus sp. CGMCC 9967. It belongs to d-3-phosphoglycerate dehydrogenase (PGDH) subfamily of 2-hydroxy acid dehydrogenase superfamily. LaPPR was stable at pH 6.5 and 30°C, with a half-life of 152h. LaPPR has a substrate preference towards aromatic to aliphatic keto acids, and various keto acids could be reduced into d-hydroxy acids with excellent enantioselectivity (>99%). By construction the coexpression system with glucose dehydrogenase, as much as 100gL−1 phenylpyruvic acid was asymmetrically reduced into d-phenyllactic acid with 91.3% isolation yield and 243gL−1d−1 productivity. The results suggest that LaPPR is a promising biocatalyst for the efficient synthesis of optically pure d-phenyllactic acid.

Phenyllactic acid production by simultaneous saccharification and fermentation of pretreated sorghum bagasse

Dilute acid-pretreated sorghum bagasse, which was predominantly composed of glucan (59%) and xylose (7.2%), was used as a lignocellulosic feedstock for d-phenyllactic acid (PhLA) production by a recombinant Escherichia coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens. During fermentation with enzymatic hydrolysate of sorghum bagasse as a carbon source, the PhLA yield was reduced by 35% compared to filter paper hydrolysate, and metabolomics analysis revealed that NAD(P)H regeneration and intracellular levels of erythrose-4-phosphate and phosphoenolpyruvate for PhLA biosynthesis markedly reduced. Compared to separate hydrolysis and fermentation (SHF) with sorghum bagasse hydrolysate, simultaneous saccharification and fermentation (SSF) of sorghum bagasse under glucose limitation conditions yielded 4.8-fold more PhLA with less accumulation of eluted components, including p-coumaric acid and aldehydes, which inhibited PhLA fermentation. These results suggest that gradual enzymatic hydrolysis during SSF enhances PhLA production under glucose limitation and reduces the accumulation of fermentation inhibitors, collectively leading to increased PhLA yield.

Production of natural antimicrobial compound D-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active D-lactate dehydrogenase.

Phenyllactic acid (PLA) is an antimicrobial compound naturally synthesized in various fermented foods and its D-form of PLA is known to be more active than the L-isomer. In this study, Leuconostoc mesenteroides ATCC 8293 cells, elaborating D-lactate dehydrogenase (D-ldh) were used to produce D-PLA from phenylpyruvic acid (PPA). When cultured in the presence of PPA (≤50 mmol l(-1)), growing cells produced a maximum yield of 35 mmol l(-1) of D-PLA, and the yields were between 75·2 and 83·3%. Higher conversion yields were obtained at pH 6·0-7·0 when growing cells were used, while the optimum pH range was broader for resting cells. The time required for the complete conversion of PPA into PLA could be shortened to 3 h using resting cells. D-ldh, an enzyme encoded by the LEUM_1756 gene of Leuc. mesenteroides ATCC 8293, was found to be responsible for the conversion of PPA into PLA. The Km and kcat values of the enzyme for PPA were found to be 15·4 mmol l(-1) and 5645 s(-1), respectively. The conditions required for the efficient production of D-PLA were optimized for both growing and resting cells of Leuc. mesenteroides, with special emphasis on achieving high stereoselectivity and conversion yield. Significance and impact of the study: This is the first study on the production of D-phenyllactic acid, which is a natural antimicrobial compound, from phenylpyruvate using Leuconostoc mesenteroides cells. The strain, ATCC 8293, that was used in the study, possesses high stereoselectivity and delivers a high yield. Therefore, it might be a promising candidate for use in large-scale production facilities and in fermented foods.