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Biotechnology For Biofuels and... May 2022Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to...
BACKGROUND
Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene 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 pathways, Kp-IpdC has an 2.8-fold lower K for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG 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 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of 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.
PubMed: 35501883
DOI: 10.1186/s13068-022-02144-8 -
RSC Advances Sep 2021Indole-3-acetic acid (IAA) plays an important role in the growth and development of plants. In this study, a series of predominant strains were isolated and identified...
Indole-3-acetic acid (IAA) plays an important role in the growth and development of plants. In this study, a series of predominant strains were isolated and identified as sp. with remarkable IAA-producing capabilities. The IAA-producing strains are mainly tryptophan-dependent and have significantly high yields of IAA (3477 μg mL and 3378 μg mL). The gene encoding indole-3-pyruvate decarboxylase was identified by genomic analysis and RT-qPCR analysis, indicating the involvement of the indole-3-pyruvic acid (IPyA) pathway of IAA biosynthesis. The IPyA pathway was also confirmed by the intermediate assay. The IAA product of microbial metabolites was isolated, purified and characterized. These microbes exhibiting IAA production significantly promoted the growth of maize, increasing root length, plant height, fresh weight and dry weight. Thus, sp. with high IAA production has great prospects in agricultural and industrial applications.
PubMed: 35496854
DOI: 10.1039/d1ra05659j -
Frontiers in Plant Science 2022Litchi is a highly perishable fruit. Ripe litchi fruit loses quality quickly as they hang on tree, giving a very short hanging life and thus harvest period. This study...
Litchi is a highly perishable fruit. Ripe litchi fruit loses quality quickly as they hang on tree, giving a very short hanging life and thus harvest period. This study attempted to explore the roles of cytokinin in regulating fruit ripening and senescence of litchi and examine the possibility of applying cytokinin in "on-tree storage" of the fruit. Exogenous cytokinin, forchlorfenuron (CPPU), was applied at 20 mg L 7 weeks after full bloom on litchi ( cv. Feizixiao) fruit clusters. Color parameters, chlorophylls, anthocyanins, fruit and fruit part weights, total soluble solutes (TSSs), soluble sugars, organic acids, non-anthocyanin flavonoids, ethanol, and also CPPU residue in fruit were traced. CPPU residue was higher but decreased faster in the pericarp than in the aril, where it maintained < 10 μg kg. CPPU had no significant effect on fruit weight but tended to increase pericarp weight. The treatment suppressed chlorophyll loss and anthocyanin accumulation in the pericarp, increased non-anthocyanin flavonoids in the aril, but had no significant effects on non-anthocyanin flavonoids in the pericarp and total sugar and organic acids in the aril. As the commercially ripe fruit hanged on tree, TSSs, total sugar, and sucrose decreased with ethanol and acetic acid accumulation in the aril. CPPU significantly suppressed the loss of sucrose and total sugar and the accumulation of ethanol and acetic acid in the aril and inhibited malondialdehyde accumulation in the pericarp of the overripe fruit. Soluble invertase, alcohol dehydrogenase, and pyruvate decarboxylase (PDC) activity and gene expression in the aril were downregulated by CPPU. The results suggest that cytokinin partially suppresses the ripening process in litchi and is effective to slow quality loss in the overripe fruit on tree.
PubMed: 35310679
DOI: 10.3389/fpls.2022.829635 -
Scientific Reports Mar 2022Protein tyrosine nitration (PTN), in which tyrosine (Tyr) residues on proteins are converted into 3-nitrotyrosine (NT), is one of the post-translational modifications...
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.
Topics: Ethanol; Fermentation; Nitrites; Proteomics; Pyruvate Decarboxylase; Reactive Nitrogen Species; Saccharomyces cerevisiae; Tyrosine
PubMed: 35304512
DOI: 10.1038/s41598-022-08568-4 -
Journal of Fungi (Basel, Switzerland) Feb 2022The rice blast fungus has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is...
The rice blast fungus has been known to produce the phytohormone auxin/IAA from its hyphae and conidia, but the detailed biological function and biosynthesis pathway is largely unknown. By sequence homology, we identified a complete indole-3-pyruvic acid (IPA)-based IAA biosynthesis pathway in , consisting of the tryptophan aminotransferase (MoTam1) and the indole-3-pyruvate decarboxylase (MoIpd1). In comparison to the wild type, IAA production was significantly reduced in the Δ mutant, and further reduced in the Δ mutant. Correspondingly, mycelial growth, conidiation, and pathogenicity were defective in the Δ and the Δ mutants to various degrees. Targeted metabolomics analysis further confirmed the presence of a functional IPA pathway, catalyzed by MoIpd1, which contributes to IAA/auxin production in . Furthermore, the well-established IAA biosynthesis inhibitor, yucasin, suppressed mycelial growth, conidiation, and pathogenicity in . Overall, this study identified an IPA-dependent IAA synthesis pathway crucial for mycelial growth and pathogenic development.
PubMed: 35205962
DOI: 10.3390/jof8020208 -
Biotechnology Reports (Amsterdam,... Mar 2022was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather...
was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather than via glyoxylate shunt, implying oxaloacetate formation from pyruvate and artificial channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation, was implemented. The main fumarases were inactivated in the core strain MSG1.0 (∆, ∆, ∆, ∆, ∆, P-, P-) by the deletion of the , and genes. The gene was expressed in the strain to ensure pyruvate to oxaloacetate conversion. The gene was expressed to enable succinate semialdehyde formation. The resulting strain was able to convert glucose to fumaric acid with a yield of 0.86 mol/mol, amounting to 86% of the theoretical maximum. The results demonstrated the high potential of the implemented strategy for development of efficient strains for bio-based fumaric acid production.
PubMed: 35145886
DOI: 10.1016/j.btre.2022.e00703 -
Biology Jan 2022Waterlogging is one of the serious abiotic stresses that inhibits crop growth and reduces productivity. Therefore, investigating efficient waterlogging mitigation...
Waterlogging is one of the serious abiotic stresses that inhibits crop growth and reduces productivity. Therefore, investigating efficient waterlogging mitigation measures has both theoretical and practical significance. The objectives of the present research were to examine the efficiency of melatonin and KNO seed soaking and foliar application on alleviating the waterlogging inhibited growth performance of maize seedlings. In this study, 100 µM melatonin and different levels (0.25, 0.50 and 0.75 g) of potassium nitrate (KNO) were used in seed soaking and foliar applications. For foliar application, treatments were applied at the 7th leaf stage one week after the imposition of waterlogging stress. The results showed that melatonin with KNO significantly improved the plant growth and biochemical parameters of maize seedlings under waterlogging stress conditions. However, the application of melatonin with KNO treatments increased plant growth characteristics, chlorophyll content, and the net photosynthetic rate at a variable rate under waterlogging stress. Furthermore, melatonin with KNO treatments significantly reduced the accumulation of hydrogen peroxide (HO) and malondialdehyde (MDA), and it decreased the activity of pyruvate decarboxylase and alcohol dehydrogenase, while increasing enzymatic activities and soluble protein content of maize seedlings under waterlogging stress conditions. Overall, our results indicated that seed soaking with 100 µM melatonin and 0.50 g KNO was the most effective treatment that significantly improved the plant growth characteristics, chlorophyll content, photosynthetic rate, and enzymatic activity of maize seedling under waterlogging conditions.
PubMed: 35053096
DOI: 10.3390/biology11010099 -
Journal of Experimental Botany Mar 2022C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait...
C4 photosynthesis concentrates CO2 around Rubisco in the bundle sheath, favouring carboxylation over oxygenation and decreasing photorespiration. This complex trait evolved independently in >60 angiosperm lineages. Its evolution can be investigated in genera such as Flaveria (Asteraceae) that contain species representing intermediate stages between C3 and C4 photosynthesis. Previous studies have indicated that the first major change in metabolism probably involved relocation of glycine decarboxylase and photorespiratory CO2 release to the bundle sheath and establishment of intercellular shuttles to maintain nitrogen stoichiometry. This was followed by selection for a CO2-concentrating cycle between phosphoenolpyruvate carboxylase in the mesophyll and decarboxylases in the bundle sheath, and relocation of Rubisco to the latter. We have profiled 52 metabolites in nine Flaveria species and analysed 13CO2 labelling patterns for four species. Our results point to operation of multiple shuttles, including movement of aspartate in C3-C4 intermediates and a switch towards a malate/pyruvate shuttle in C4-like species. The malate/pyruvate shuttle increases from C4-like to complete C4 species, accompanied by a rise in ancillary organic acid pools. Our findings support current models and uncover further modifications of metabolism along the evolutionary path to C4 photosynthesis in the genus Flaveria.
Topics: Flaveria; Glycine Dehydrogenase (Decarboxylating); Metabolome; Photosynthesis; Ribulose-Bisphosphate Carboxylase
PubMed: 34910813
DOI: 10.1093/jxb/erab540 -
Anais Da Academia Brasileira de Ciencias 2021The kinetic profiles of Candida tropicalis TISTR 5306 cultivation based on modified yeast-malt (MYM), assorted grade fresh longan juice (AsgLG) and longan solid waste...
The kinetic profiles of Candida tropicalis TISTR 5306 cultivation based on modified yeast-malt (MYM), assorted grade fresh longan juice (AsgLG) and longan solid waste extract (LSWE) medium were evaluated in 1 l batch mode. The highest ethanol concentration level (25.5 ± 0.8 g/l) and ethanol yield - Yp/s of 0.491 ± 0.017 g ethanol/g consumed substrate, dried biomass concentration level (9.44 ± 0.05 g/l) and dried biomass yield - Yp/s of 0.533 ± 0.170 g dried biomass/g consumed substrate, specific pyruvate decarboxylase (PDC) activity (0.037 ± 0.003 U/mg protein) were achieved (p ≤ 0.05) in AsgLG medium. Scores ranking strategy were employed and AsgLG medium was subsequently selected with in the highest total score (p ≤ 0.05) of 698 ± 7 at 48 h. The cultivation in fed-batch mode with three rounds of pulse feeding (PF) in 1 l AsgLG medium was carried out. The apparent highest ethanol and dried biomass concentration levels with corresponding yields relative to time zero were (28.3 ± 0.5 g/l, 0.482 ± 0.012 g/g) at 120 h of PF2 and (9.39 ± 0.04 g/l, 0.110 ± 0.001 g/g) at 192 h of PF3. The maximum specific PDC activity was 0.057 ± 0.006 U/mg protein during PF1 feeding.
Topics: Biomass; Candida tropicalis; Ethanol; Fermentation; Kinetics; Sapindaceae
PubMed: 34877969
DOI: 10.1590/0001-3765202120200220 -
Frontiers in Plant Science 2021Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon...
Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower and genes ( and were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in and , where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.
PubMed: 34868183
DOI: 10.3389/fpls.2021.781917