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Molecules (Basel, Switzerland) Jul 2023Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of...
Many enzymes have latent activities that can be used in the conversion of non-natural reactants for novel organic conversions. A classic example is the conversion of benzaldehyde to a phenylacetyl carbinol, a precursor for ephedrine manufacture. It is often tacitly assumed that purified enzymes are more promising catalysts than whole cells, despite the lower cost and easier maintenance of the latter. Competing substrates inside the cell have been known to elicit currently hard-to-predict selectivities that are not easily measured inside the living cell. We employ NMR spectroscopic assays to rationally combine isomers for selective reactions in commercial . This approach uses internal competition between alternative pathways of aldehyde clearance in yeast, leading to altered selectivities compared to catalysis with the purified enzyme. In this manner, 4-fluorobenzyl alcohol and 2-fluorophenylacetyl carbinol can be formed with selectivities in the order of 90%. Modification of the cellular redox state can be used to tune product composition further. Hyperpolarized NMR shows that the cellular reaction and pathway usage are affected by the xenochemical. Overall, we find that the rational construction of ternary or more complex substrate mixtures can be used for in-cell NMR spectroscopy to optimize the upgrading of similar xenochemicals to dissimilar products with cheap whole-cell catalysts.
Topics: Methanol; Saccharomyces cerevisiae; Catalysis; Alcohols; Ephedrine
PubMed: 37446819
DOI: 10.3390/molecules28135157 -
l-Lactic Acid Production via Sustainable Neutralizer-Free Route by Engineering Acid-Tolerant Yeast .Journal of Agricultural and Food... Jul 2023l-Lactic acid (l-LA) is a platform chemical obtained via microbial fermentation at a near-neutral pH value. Large amounts of neutralizers are required during this...
l-Lactic acid (l-LA) is a platform chemical obtained via microbial fermentation at a near-neutral pH value. Large amounts of neutralizers are required during this process, which increases the production costs in downstream processing as well as environmental burden. To address this challenge, an acid-tolerant yeast E1 was isolated and metabolically engineered to produce l-LA without neutralizers. The genome of strain E1 was sequenced and a CRISPR-Cas9 system was developed in this newly isolated strain. Subsequently, the gene encoding pyruvate decarboxylase () was knocked out to subdue ethanol formation. Furthermore, the l-lactate dehydrogenase gene from 2-6 and the codon-optimized gene from were introduced into E1 chromosome to redirect the ethanol fermentation pathway to l-LA production. Deletion of the () gene further increased the optical purity of l-LA. After optimizing fermentation conditions, the maximum titer of l-LA in the 5 L fermenter reached 74.57 g/L without any neutralizers, with an optical purity of 100% and a maximum yield of 0.93 g/g glucose. This is the first report of optically pure l-LA production without neutralizers and the engineered acid-tolerant yeast paves the way for the sustainable production of l-LA via a green route.
Topics: Animals; Cattle; Saccharomyces cerevisiae; Lactic Acid; Acids; Pichia; Fermentation; Ethanol
PubMed: 37439413
DOI: 10.1021/acs.jafc.3c03163 -
Applied Microbiology and Biotechnology Aug 2023Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies...
Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.
Topics: Lactic Acid; Saccharomyces cerevisiae; Kluyveromyces; L-Lactate Dehydrogenase; Glucose; Pyruvate Decarboxylase; Hydrogen-Ion Concentration; Fermentation
PubMed: 37405435
DOI: 10.1007/s00253-023-12658-2 -
Plant Physiology and Biochemistry : PPB Aug 2023Hypoxic stress due to submergence is a serious threat to the growth and development of maize. WRKY transcription factors are significant regulators of plant responses to...
Hypoxic stress due to submergence is a serious threat to the growth and development of maize. WRKY transcription factors are significant regulators of plant responses to various abiotic and biotic stresses. Nevertheless, their function and regulatory mechanisms in the resistance of maize to submergence stress remain unclear. Here we report the cloning of a maize WRKY transcription factor gene, ZmWRKY70, transcripts of which accumulate under submergence stress in maize seedlings. Subcellular localization analysis and yeast transcriptional activation assay indicated that ZmWRKY70 was localized in the nucleus and had transcriptional activation activity. Heterologous overexpression of ZmWRKY70 in Arabidopsis increased the tolerance of seeds and seedlings to submergence stress by upregulating the transcripts of several key genes involved in anaerobic respiration, such as group VII ethylene-responsive factor (ERFVII) (AtRAP2.2), alcohol dehydrogenase (AtADH1), pyruvate decarboxylase (AtPDC1/2), and sucrose synthase (AtSUS4), under submergence conditions. Moreover, the overexpression of ZmWRKY70 in maize mesophyll protoplasts enhanced the expression of ZmERFVII members (ZmERF148, ZmERF179, and ZmERF193), ZmADH1, ZmPDC2/3, and ZmSUS1. Yeast one-hybrid and dual-luciferase activity assays further confirmed that ZmWRKY70 enhanced the expression of ZmERF148 by binding to the W box motif located in the promoter region of ZmERF148. Together, these results indicate that ZmWRKY70 plays a significant role in tolerance of submergence stress. This work provides a theoretical basis, and suggests excellent genes, for biotechnological breeding to improve the tolerance of maize to submergence through the regulation of ZmWRKY genes.
Topics: Arabidopsis; Zea mays; Saccharomyces cerevisiae; Plant Breeding; Transcription Factors; Stress, Physiological; Seedlings; Gene Expression Regulation, Plant; Plants, Genetically Modified; Plant Proteins
PubMed: 37364509
DOI: 10.1016/j.plaphy.2023.107861 -
BMC Plant Biology Jun 2023Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is...
BACKGROUND
Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown.
RESULTS
The transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress.
CONCLUSIONS
Regulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.
Topics: Gene Expression Profiling; Transcriptome; Carbohydrate Metabolism; Stress, Physiological; Ethylenes; Gene Expression Regulation, Plant
PubMed: 37344795
DOI: 10.1186/s12870-023-04334-4 -
Frontiers in Microbiology 2023Cyanobacteria are an excellent microbial photosynthetic platform for sustainable carbon dioxide fixation. One bottleneck to limit its application is that the natural...
Cyanobacteria are an excellent microbial photosynthetic platform for sustainable carbon dioxide fixation. One bottleneck to limit its application is that the natural carbon flow pathway almost transfers CO to glycogen/biomass other than designed biofuels such as ethanol. Here, we used engineered sp. PCC 6803 to explore CO-to-ethanol potential under atmospheric environment. First, we investigated the effects of two heterologous genes (pyruvate decarboxylase and alcohol dehydrogenase) on ethanol biosynthesis and optimized their promoter. Furthermore, the main carbon flow of the ethanol pathway was strengthened by blocking glycogen storage and pyruvate-to-phosphoenolpyruvate backflow. To recycle carbon atoms that escaped from the tricarboxylic acid cycle, malate was artificially guided back into pyruvate, which also created NADPH balance and promoted acetaldehyde conversion into ethanol. Impressively, we achieved high-rate ethanol production (248 mg/L/day at early 4 days) by fixing atmospheric CO. Thus, this study exhibits the proof-of-concept that rewiring carbon flow strategies could provide an efficient cyanobacterial platform for sustainable biofuel production from atmospheric CO.
PubMed: 37323905
DOI: 10.3389/fmicb.2023.1211004 -
Plant Physiology Sep 2023Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that...
Root growth in maize (Zea mays L.) is regulated by the activity of the quiescent center (QC) stem cells located within the root apical meristem. Here, we show that despite being highly hypoxic under normal oxygen tension, QC stem cells are vulnerable to hypoxic stress, which causes their degradation with subsequent inhibition of root growth. Under low oxygen, QC stem cells became depleted of starch and soluble sugars and exhibited reliance on glycolytic fermentation with the impairment of the TCA cycle through the depressed activity of several enzymes, including pyruvate dehydrogenase (PDH). This finding suggests that carbohydrate delivery from the shoot might be insufficient to meet the metabolic demand of QC stem cells during stress. Some metabolic changes characteristic of the hypoxic response in mature root cells were not observed in the QC. Hypoxia-responsive genes, such as PYRUVATE DECARBOXYLASE (PDC) and ALCOHOL DEHYDROGENASE (ADH), were not activated in response to hypoxia, despite an increase in ADH activity. Increases in phosphoenolpyruvate (PEP) with little change in steady-state levels of succinate were also atypical responses to low-oxygen tensions. Overexpression of PHYTOGLOBIN 1 (ZmPgb1.1) preserved the functionality of the QC stem cells during stress. The QC stem cell preservation was underpinned by extensive metabolic rewiring centered around activation of the TCA cycle and retention of carbohydrate storage products, denoting a more efficient energy production and diminished demand for carbohydrates under conditions where nutrient transport may be limiting. Overall, this study provides an overview of metabolic responses occurring in plant stem cells during oxygen deficiency.
Topics: Plant Roots; Oxygen; Meristem; Stem Cells; Hypoxia; Carbohydrates
PubMed: 37311198
DOI: 10.1093/plphys/kiad344 -
Foods (Basel, Switzerland) May 2023Ellis & Halsted is the pathogen causing black rot in sweet potatoes that can lead to flavor change and toxin release. This study detected the volatile organic compounds...
Identifying Early-Stage Changes in Volatile Organic Compounds of Ellis & Halsted-Infected Sweet Potatoes ( L. Lam) Using Headspace Gas Chromatography-Ion Mobility Spectrometry.
Ellis & Halsted is the pathogen causing black rot in sweet potatoes that can lead to flavor change and toxin release. This study detected the volatile organic compounds (VOCs) of -infected sweet potatoes in the early stages using headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 55 VOCs were identified, including aldehydes, alcohols, esters, ketones, and others. The content of aldehydes and ketones showed a decreasing trend, while alcohols and esters showed an increasing trend. An increase in infection time elevated the content of malondialdehyde (MDA) and pyruvate, while the starch content decreased, the content of soluble protein initially increased, then decreased, and the activities of lipoxygenase (LOX), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and phenylalanine ammonia-lyase (PAL) increased. The changes in VOCs were closely related to the content of MDA, starch, pyruvate, and the activities of LOX, PDC, ADH, and PAL. Sweet potatoes showed a good discrimination effect by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) from 0 to 72 h. Twenty-five differential VOCs could be used as early-stage characteristic compounds of -infected sweet potatoes for early disease monitoring.
PubMed: 37297466
DOI: 10.3390/foods12112224 -
PloS One 2023Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially...
Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.
Topics: Saccharomyces cerevisiae; Candida glabrata; Transcription Factors; Fungal Proteins; Pyruvate Decarboxylase; Thiamine; Carboxy-Lyases; Promoter Regions, Genetic; Intrinsically Disordered Proteins; Gene Expression Regulation, Fungal
PubMed: 37285346
DOI: 10.1371/journal.pone.0286744 -
Life (Basel, Switzerland) Apr 2023Mulberry (), a widely distributed economic plant, can withstand long-term flooding stress. However, the regulatory gene network underlying this tolerance is unknown. In...
Mulberry (), a widely distributed economic plant, can withstand long-term flooding stress. However, the regulatory gene network underlying this tolerance is unknown. In the present study, mulberry plants were subjected to submergence stress. Subsequently, mulberry leaves were collected to perform quantitative reverse-transcription PCR (qRT-PCR) and transcriptome analysis. Genes encoding ascorbate peroxidase and glutathione S-transferase were significantly upregulated after submergence stress, indicating that they could protect the mulberry plant from flood damage by mediating ROS homeostasis. Genes that regulate starch and sucrose metabolism; genes encoding pyruvate kinase, alcohol dehydrogenase, and pyruvate decarboxylase (enzymes involved in glycolysis and ethanol fermentation); and genes encoding malate dehydrogenase and ATPase (enzymes involved in the TCA cycle) were also obviously upregulated. Hence, these genes likely played a key role in mitigating energy shortage during flooding stress. In addition, genes associated with ethylene, cytokinin, abscisic acid, and MAPK signaling; genes involved in phenylpropanoid biosynthesis; and transcription factor genes also showed upregulation under flooding stress in mulberry plants. These results provide further insights into the adaptation mechanisms and genetics of submergence tolerance in mulberry plants and could aid in the molecular breeding of these plants.
PubMed: 37240733
DOI: 10.3390/life13051087