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.

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A novel rat homolog of the Saccharomyces cerevisiae ubiquitin-conjugating enzymes UBC4 and UBC5 with distinct biochemical features is induced during spermatogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4064 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4064-4072 ◽

Cited By ~ 40

Author(s):

S S Wing ◽

N Bédard ◽

C Morales ◽

P Hingamp ◽

J Trasler

Keyword(s):

Saccharomyces Cerevisiae ◽

Sequence Similarity ◽

Amino Acid Identity ◽

Cellular Function ◽

Rat Tissues ◽

Regulation Of Expression ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

First Time

The Saccharomyces cerevisiae ubiquitin-conjugating enzymes (E2s) UBC4 and UBC5 are essential for degradation of short-lived and abnormal proteins. We previously identified rat cDNAs encoding two E2s with strong sequence similarity to UBC4 and UBC5. These E2 isoforms are widely expressed in rat tissues, consistent with a fundamental cellular function for these E2s. We now report a new isoform, 8A, which despite having >91% amino acid identity with the other isoforms, shows several novel features. Expression of the 8A isoform appears restricted to the testis, is absent in early life, but is induced during puberty. Hypophysectomy reduced expression of the 8A isoform. In situ hybridization studies indicated that 8A mRNA is expressed mainly in round spermatids. Immunoblot analyses showed that 8A protein is found not only in subfractions of germ cells enriched in round spermatids but also in subfractions containing residual bodies extruded from more mature elongated spermatids, indicating that the protein possesses a longer half-life than the mRNA. Unlike all previously identified mammalian and plant homologs of S. cerevisiae UBC4, which possess a basic pI, the 8A isoform is unique in possessing an acidic pI. The small differences in sequence between the 8A isoform and other rat isoforms conferred differences in biochemical function. The 8A isoform was less effective than an isoform with a basic pI or ineffective in conjugating ubiquitin to certain fractions of testis proteins. Thus, although multiple isoforms of a specific E2 may exist to ensure performance of a critical cellular function, our data demonstrate, for the first time, that multiple genes also permit highly specialized regulation of expression of specific isoforms and that subtle differences in E2 primary structure can dictate conjugation of ubiquitin to different subsets of cellular proteins.

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Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.92.3.790 ◽

1982 ◽

Vol 92 (3) ◽

pp. 790-794 ◽

Cited By ~ 25

Author(s):

M Nagy ◽

J Laporte ◽

B Penverne ◽

G Hervé

Keyword(s):

Saccharomyces Cerevisiae ◽

Feedback Inhibition ◽

Nuclear Accumulation ◽

Lead Phosphate ◽

Carbamoylphosphate Synthetase ◽

Aspartate Carbamoyltransferase ◽

Encoding Gene ◽

Atcase Activity ◽

Cytochemical Technique

The cytochemical technique using the in situ precipitation of orthophosphate ions liberated specifically by the aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) reaction indicated that in Saccharomyces cerevisiae this enzyme is confined to the nucleus. This observation is in accordance with the result reported by Bernhardt and Davis (1972), Proc. Natl. Acad. Sci. U. S. A. 69:1868-1872) on Neurospora crassa. The nuclear compartmentation was also observed in a mutant strain lacking proteinase B activity. This finding indicates that this proteinase is not involved in the nuclear accumulation of ATCase, and that the activity observed in the nucleus corresponds to the multifunctional form associated with the uracil path-specific carbamoylphosphate synthetase and sensitive to feedback inhibition by UTP. In a ura2 strain transformed by nonintegrated pFL1 plasmids bearing the URA2-ATCase activity encoding gene, the lead phosphate precipitate was observed predominantly in the cytoplasm. This finding enhances the reliability of the technique used by eliminating the possibility of an artifactual displacement of an originally cytoplasmic reaction product during the preparation of the material for electron microscopy. On the other hand, nuclei isolated under hypoosmotic conditions do not exhibit the ATCase activity that is recovered in the cytosolic fractions after differential centrifugation of the lysate in Percoll gradient. A release of the protein from the nuclei during the lysis step, consistent with its nucleoplasmic localization, is postulated.

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Probing the Dynamic Self-Assembly Behaviour of Photoswitchable Wormlike Micelles in Real Time

10.26434/chemrxiv.7098695 ◽

2018 ◽

Author(s):

Elaine A. Kelly ◽

Judith E. Houston ◽

Rachel Evans

Keyword(s):

Real Time ◽

Absorption Spectroscopy ◽

Self Assembly ◽

Uv Light ◽

Wormlike Micelles ◽

Tetraethylene Glycol ◽

Light Illumination ◽

First Time ◽

Dependent Processes

Understanding the dynamic self-assembly behaviour of azobenzene photosurfactants (AzoPS) is crucial to advance their use in controlled release applications such asdrug delivery and micellar catalysis. Currently, their behaviour in the equilibrium cis-and trans-photostationary states is more widely understood than during the photoisomerisation process itself. Here, we investigate the time-dependent self-assembly of the different photoisomers of a model neutral AzoPS, <a>tetraethylene glycol mono(4′,4-octyloxy,octyl-azobenzene) </a>(C8AzoOC8E4) using small-angle neutron scattering (SANS). We show that the incorporation of in-situUV-Vis absorption spectroscopy with SANS allows the scattering profile, and hence micelle shape, to be correlated with the extent of photoisomerisation in real-time. It was observed that C8AzoOC8E4could switch between wormlike micelles (transnative state) and fractal aggregates (under UV light), with changes in the self-assembled structure arising concurrently with changes in the absorption spectrum. Wormlike micelles could be recovered within 60 seconds of blue light illumination. To the best of our knowledge, this is the first time the degree of AzoPS photoisomerisation has been tracked in-situthrough combined UV-Vis absorption spectroscopy-SANS measurements. This technique could be widely used to gain mechanistic and kinetic insights into light-dependent processes that are reliant on self-assembly.

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The Total Synthesis of Rhabdastrellic Acid A

10.26434/chemrxiv.7479944 ◽

2018 ◽

Author(s):

Yaroslav Boyko ◽

Christopher Huck ◽

David Sarlah

Keyword(s):

Total Synthesis ◽

Late Stage ◽

Cross Coupling ◽

Side Chains ◽

General Strategy ◽

Modular Approach ◽

Linear Sequence ◽

Generating Sequence ◽

First Time

<div>The first total synthesis of rhabdastrellic acid A, a highly cytotoxic isomalabaricane triterpenoid, has been accomplished in a linear sequence of 14 steps from commercial geranylacetone. The prominently strained trans-syn-trans-perhydrobenz[e]indene core characteristic of the isomalabaricanes is efficiently accessed in a selective manner for the first time through a rapid, complexity-generating sequence incorporating a reductive radical polyene cyclization, an unprecedented oxidative Rautenstrauch cycloisomerization, and umpolung 𝛼-substitution of a p-toluenesulfonylhydrazone with in situ reductive transposition. A late-stage cross-coupling in concert with a modular approach to polyunsaturated side chains renders this a general strategy for the synthesis of numerous family members of these synthetically challenging and hitherto inaccessible marine triterpenoids.</div>

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Transcriptome Analysis Reveals a Promotion of Carotenoid Production by Copper Ions in Recombinant Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms9020233 ◽

2021 ◽

Vol 9 (2) ◽

pp. 233

Author(s):

Buli Su ◽

Anzhang Li ◽

Ming-Rong Deng ◽

Honghui Zhu

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptome Analysis ◽

Gene Expression Analysis ◽

Target Genes ◽

Copper Ions ◽

Carotenoid Production ◽

Carotenoid Accumulation ◽

Nutritional Components ◽

Differential Gene ◽

First Time

We previously constructed a Saccharomyces cerevisiae carotenoid producer BL03-D-4 which produced much more carotenoid in YPM (modified YPD) media than YPD media. In this study, the impacts of nutritional components on carotenoid accumulation of BL03-D-4 were investigated. When using YPM media, the carotenoid yield was increased 10-fold compared to using the YPD media. To elucidate the hidden mechanism, a transcriptome analysis was performed and showed that 464 genes changed significantly in YPM media. Furthermore, inspired by the differential gene expression analysis which indicated that ADY2, HES1, and CUP1 showed the most remarkable changes, we found that the improvement of carotenoid accumulation in YPM media was mainly due to the copper ions, since supplementation of 0.08 mM CuSO4 in YPD media could increase carotenoid yield 9.2-fold. Reverse engineering of target genes was performed and carotenoid yield could be increased 6.4-fold in YPD media through overexpression of ACE1. The present study revealed for the first time the prominent promotion of carotenoid yield by copper ions in engineered S. cerevisiae and provided a new target ACE1 for genetic engineering of S. cerevisiae for the bioproduction of carotenoids.

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Chromosomal painting of the sandpiper (Actitis macularius) detects several fissions for the Scolopacidae family (Charadriiformes)

BMC Ecology and Evolution ◽

10.1186/s12862-020-01737-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Melquizedec Luiz Silva Pinheiro ◽

Cleusa Yoshiko Nagamachi ◽

Talita Fernanda Augusto Ribas ◽

Cristovam Guerreiro Diniz ◽

Patricia Caroline Mary O´Brien ◽

...

Keyword(s):

Chromosome Painting ◽

Diploid Number ◽

Chromosomal Evolution ◽

Long Distance ◽

Chromosomal Painting ◽

Cytogenetic Studies ◽

Burhinus Oedicnemus ◽

Clear Idea ◽

First Time

Abstract Background The Scolopacidae family (Suborder Scolopaci, Charadriiformes) is composed of sandpipers and snipes; these birds are long-distance migrants that show great diversity in their behavior and habitat use. Cytogenetic studies in the Scolopacidae family show the highest diploid numbers for order Charadriiformes. This work analyzes for the first time the karyotype of Actitis macularius by classic cytogenetics and chromosome painting. Results The species has a diploid number of 92, composed mostly of telocentric pairs. This high 2n is greater than the proposed 80 for the avian ancestral putative karyotype (a common feature among Scolopaci), suggesting that fission rearrangements have formed smaller macrochromosomes and microchromosomes. Fluorescence in situ hybridization using Burhinus oedicnemus whole chromosome probes confirmed the fissions in pairs 1, 2, 3, 4 and 6 of macrochromosomes. Conclusion Comparative analysis with other species of Charadriiformes studied by chromosome painting together with the molecular phylogenies for the order allowed us to raise hypotheses about the chromosomal evolution in suborder Scolopaci. From this, we can establish a clear idea of how chromosomal evolution occurred in this suborder.

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Stretchable supercapacitor at −30 °C

Energy & Environmental Science ◽

10.1039/d0ee04066e ◽

2021 ◽

Author(s):

Xuting Jin ◽

Li Song ◽

Hongsheng Yang ◽

Chunlong Dai ◽

Yukun Xiao ◽

...

Keyword(s):

First Time

A stretchable supercapacitor at −30 °C was developed for the first time by in situ growth of polyaniline onto the newly-designed anti-freezing organohydrogel polyelectrolyte.

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Direct TEM observation of the “acanthite α-Ag2S–argentite β-Ag2S” phase transition in a silver sulfide nanoparticle

Nanoscale Advances ◽

10.1039/c8na00347e ◽

2019 ◽

Vol 1 (4) ◽

pp. 1581-1588 ◽

Cited By ~ 6

Author(s):

S. I. Sadovnikov ◽

E. Yu. Gerasimov

Keyword(s):

Electron Microscopy ◽

Phase Transition ◽

Transmission Electron Microscopy ◽

Silver Sulfide ◽

Transmission Electron ◽

Silver Sulfide Nanoparticles ◽

Microscopy Method ◽

Electron Microscopy Method ◽

First Time

For the first time, the α-Ag2S (acanthite)–β-Ag2S (argentite) phase transition in a single silver sulfide nanoparticles has been observed in situ using a high-resolution transmission electron microscopy method in real time.

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Microbial N2O consumption in and above marine N2O production hotspots

The ISME Journal ◽

10.1038/s41396-020-00861-2 ◽

2020 ◽

Author(s):

Xin Sun ◽

Amal Jayakumar ◽

John C. Tracey ◽

Elizabeth Wallace ◽

Colette L. Kelly ◽

...

Keyword(s):

Kinetic Parameters ◽

Substrate Affinity ◽

N2o Production ◽

Anoxic Waters ◽

Production And Consumption ◽

Consumption Rates ◽

Anoxic Zones ◽

First Time ◽

Microbial N

AbstractThe ocean is a net source of N2O, a potent greenhouse gas and ozone-depleting agent. However, the removal of N2O via microbial N2O consumption is poorly constrained and rate measurements have been restricted to anoxic waters. Here we expand N2O consumption measurements from anoxic zones to the sharp oxygen gradient above them, and experimentally determine kinetic parameters in both oxic and anoxic seawater for the first time. We find that the substrate affinity, O2 tolerance, and community composition of N2O-consuming microbes in oxic waters differ from those in the underlying anoxic layers. Kinetic parameters determined here are used to model in situ N2O production and consumption rates. Estimated in situ rates differ from measured rates, confirming the necessity to consider kinetics when predicting N2O cycling. Microbes from the oxic layer consume N2O under anoxic conditions at a much faster rate than microbes from anoxic zones. These experimental results are in keeping with model results which indicate that N2O consumption likely takes place above the oxygen deficient zone (ODZ). Thus, the dynamic layer with steep O2 and N2O gradients right above the ODZ is a previously ignored potential gatekeeper of N2O and should be accounted for in the marine N2O budget.

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