Molecular Mechanism of Ethanol Fermentation Inhibition via Protein Tyrosine Nitration of Pyruvate Decarboxylase by Reactive Nitrogen Species in Yeast

Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications mediated by reactive nitrogen species (RNS). Many recent studies have reported that PTN contributed to signaling systems by altering the structures and/or functions of proteins. This study aimed to investigate connections between PTN and the inhibitory effect of nitrite-derived RNS on fermentation ability using the yeast Saccharomyces cerevisiae. The results indicated that RNS inhibited the ethanol production of yeast cells with increased intracellular pyruvate content. We also found that RNS decreased the activities of pyruvate decarboxylase (PDC) as a critical enzyme involved in ethanol production. Our proteomic analysis revealed that the main PDC isozyme Pdc1 underwent the PTN modification at Tyr38, Tyr157, and Tyr344. The biochemical analysis using the recombinant purified Pdc1 enzyme indicated that PTN at Tyr157 or Tyr344 significantly reduced the Pdc1 activity. Interestingly, the substitution of Tyr157 or Tyr344 to phenylalanine, which is no longer converted into NT, recovered the ethanol production under the RNS treatment conditions. These findings suggest that nitrite impairs the fermentation ability of yeast by inhibiting the Pdc1 activity via its PTN modification at Tyr157 and Tyr344 of Pdc1.

Reduction of The Pyruvate Decarboxylase Activity Improves Isobutanol Production By Klebsiella Pneumoniae

Background Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. ipdC, annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. Results Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, high level expression of ipdC by induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling we identified 10 residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-mutants were selected for exploration. Mutants L546W and T290L showed 5.1% and 22.1% of catalytic efficiency on pyruvate, which were then expressed in K. pneumoniae for in vivo test. Isobutanol production by K. pneumoniae T290L was 25% higher than the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. Conclusions This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Process Optimization for Biosynthesis of Pyruvate Decarboxylase (PDC) and Neuberg's Ketol (PAC) from Novel Pichia Cecembences Through Response Surface Methodology using Industrial Waste as Substrate

Purpose Phenyl acetyl carbinol (PAC) is an intermediate for the synthesis of active pharmaceutical ingredients(ephedrine, pseudoephedrine, norephedrine etc.)which are used for the production of antiasthematics and decongestants. Chemical production of these APIsand extraction from plantis costly and cumbersome. Biosynthesis ofPACthrough condensation of benzaldehyde and acetaldehyde using PDC, amore effective method, is being used. These solvents can adversely inhibit PDC. Optimization of cointeraction of significant factors was done through Response surface methodology (RSM) in relatively short time.Method The effect of incubation time (8-18hrs), temperature (30-38oC), pH (4-10) and Inoculum size (4-10%,v/v)on PAC yield, sugar consumption and PDC activity was determined.PAC was quantifiedSpactrophotometerically and HPLC.All results and models were statistically analysed. PDC,produced in 5L flask using molasses as substrate, was exposed to 40mM benzaldehyde as whole cells, crude extract and partialy purified to determine its half life as residuel activity.Results PDC activity and PAC yield were 56.27 U/ml and 8.44 g/L, respectively. The yield of PAC(2.22 to 8.44g/L) was increased by 71% after process optimization through RSM with time (13hrs), temperature(33°C) and total sugar conc. (18%,v/v) as significant factors (p-values, 0.902, 0.260 and 0.247, respectively). Process design had Adj R2 0.562, R-Squared 0.770, Adeq Precision 4.888 with a uniformly distributed standard error.PDC used in the form of Pichia cecembences cells revealed higher stability towards benzaldehyde and temperature as compared to partially purified PDC.Whole cells and partially purified PDC showed half-lives of 240 and 72hrs at 4oC whereas, 33 and 28.5hrs at 25oC. PAC,purity though HPLC was 76.18%. Conclusions Time, temperature and sugar were significant factors as they increased the PAC biosynthesis.PDC from Pichia cecembences(crabtree negative;reported in other publication by same authors), as a whole cell and purified showed better half-lives at 4 and 25oC as compared to reported PDCs.Hence, it is a promising candidate for commercial production of PAC, as its PDC was stable at 4 and 25oC in presence of Benzaldehyde.

Proteomic Analysis Explores Interactions between Lactiplantibacillus plantarum and Saccharomyces cerevisiae during Sourdough Fermentation

Sourdough is a fermentation culture which is formed following metabolic activities of a multiple bacterial and fungal species on raw dough. However, little is known about the mechanism of interaction among different species involved in fermentation. In this study, Lactiplantibacillus plantarum Sx3 and Saccharomyces cerevisiae Sq7 were selected. Protein changes in sourdough, fermented with single culture (either Sx3 or Sq7) and mixed culture (both Sx3 and Sq7), were evaluated by proteomics. The results show that carbohydrate metabolism in mixed-culture-based sourdough is the most important metabolic pathway. A greater abundance of L-lactate dehydrogenase and UDP-glucose 4-epimerase that contribute to the quality of sourdough were observed in mixed-culture-based sourdough than those produced by a single culture. Calreticulin, enolase, seryl-tRNA synthetase, ribosomal protein L23, ribosomal protein L16, and ribosomal protein L5 that are needed for the stability of proteins were increased in mixed-culture-based sourdough. The abundance of some compounds which play an important role in enhancing the nutritional characteristics and flavour of sourdough (citrate synthase, aldehyde dehydrogenase, pyruvate decarboxylase, pyruvate dehydrogenase E1 and acetyl-CoA) was decreased. In summary, this approach provided new insights into the interaction between L. plantarum and S. cerevisiae in sourdough, which may serve as a base for further research into the detailed mechanism.

Fungal auxin is a quorum-based modulator of blast disease severity

Auxin is an important phytohormone regulating plant growth and development, and can also be produced by microbial pathogens including the rice-blast fungus Magnaporthe oryzae. However, the detailed biosynthesis pathway, biological function(s), and cellular distribution of such fungal auxin in M. oryzae remain largely unknown. Here, we report a sequential accumulation of intrinsic auxin in the three conidial cells, the infection structure (appressorium), and the invasive hyphae in M. oryzae. Such fungus-derived auxin was also secreted out and perceived by the host plants. A mitochondria-associated Indole-3-pyruvate decarboxylase, Ipd1, is essential for auxin/Indole-3-acetic acid biosynthesis in M. oryzae. The ipd1 mutant was defective in pathogenicity whereas overexpression of IPD1 led to enhanced virulence in rice. Chemical inhibition of fungal IAA biosynthesis, or its increase via external supplementation decreased or increased the severity of blast disease, respectively, in a dose-dependent manner. Furthermore, the IAA produced and secreted by M. oryzae governed the incidence and severity of blast disease in a quorum-dependent manner. Appressorium formation, conidial cell death critical for appressorium function, and the transcription of infection-related genes, MPG1 and INV1, directly correlated with cell density and/or IAA levels within the conidial population at the early stages of pathogenic development. Overall, our study revealed that the severity of blast disease is regulated via quorum sensing with intrinsic IAA serving as an associated signal transducer in rice blast.

Enhanced In Vitro Cascade Catalysis of Glycerol into Pyruvate and Acetoin by Integration with Dihydroxy Acid Dehydratase from Paralcaligenes ureilyticus

Recently, an in vitro enzymatic cascade was constructed to transform glycerol into the high-value platform chemical pyruvate. However, the low activity of dihydroxy acid dehydratase from Sulfolobus solfataricus (SsDHAD) limited the efficiency. In this study, the enzymatic reduction of pyruvate catalyzed by d-lactate dehydrogenase from Pseudomonas aeruginosa PAO1 was used to assay the activities of dihydroxy acid dehydratases. Dihydroxy acid dehydratase from Paralcaligenes ureilyticus (PuDHT) was identified as the most efficient candidate for glycerate dehydration. After the optimization of the catalytic temperature for the enzymatic cascade, comprising alditol oxidase from Streptomyces coelicolor A3, PuDHT, and catalase from Aspergillus niger, 20.50 ± 0.27 mM of glycerol was consumed in 4 h to produce 18.95 ± 0.97 mM of pyruvate with a productivity 12.15-fold higher than the previous report using SsDHAD. The enzymatic cascade was further coupled with the pyruvate decarboxylase from Zymomonas mobile for the production of another platform compound, acetoin. Acetoin at a concentration of 8.52 ± 0.12 mM was produced from 21.62 ± 0.19 mM of glycerol with a productivity of 1.42 ± 0.02 mM h−1.

Improvement of 2-phenylethanol production in Saccharomyces cerevisiae by evolutionary and rational metabolic engineering

2-Phenylethanol (2-PE) is a valuable aromatic compound with favorable flavors and good properties, resulting in its widespread application in the cosmetic, food and medical industries. In this study, a mutant strain, AD032, was first obtained by adaptive evolution under 2-PE stress. Then, a fusion protein from the Ehrlich pathway, composed of tyrB from Escherichia coli, kdcA from Lactococcus lactis and ADH2 from Saccharomyces cerevisiae, was constructed and expressed. As a result, 3.14 g/L 2-PE was achieved using L-phenylalanine as a precursor. To further increase 2-PE production, L-glutamate oxidase from Streptomyces overexpression was applied for the first time in our research to improve the supply of α-ketoglutarate in the transamination of 2-PE synthesis. Furthermore, we found that the disruption of the pyruvate decarboxylase encoding gene PDC5 caused an increase in 2-PE production, which has not yet been reported. Finally, assembly of the efficient metabolic modules and process optimization resulted in the strain RM27, which reached 4.02 g/L 2-PE production from 6.7 g/L L-phenylalanine without in situ product recovery. The strain RM27 produced 2-PE (0.8 mol/mol) with L-phenylalanine as a precursor, which was considerably high, and displayed manufacturing potential regarding food safety and process simplification aspects. This study suggests that innovative strategies regarding metabolic modularization provide improved prospects for 2-PE production in food exploitation.

Co-Infections by Fusarium circinatum and Phytophthora spp. on Pinus radiata: Complex Phenotypic and Molecular Interactions

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P. xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P. xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism.

Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System

Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 °C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported.