scholarly journals Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene

2021 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Yu Zhang ◽  
Tsan-Yu Chiu ◽  
Jin-Tao Zhang ◽  
Shu-Jie Wang ◽  
Shu-Wen Wang ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Rearrangement ◽  
Fold Increase ◽  
Host Strain ◽  
Target Product ◽  
Biosynthesis Pathway ◽  
Suitable Host ◽  
Enhanced Production ◽  
Condition Optimization ◽  
Heterologous Pathway

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

scholarly journals Mating of 2 Laboratory Saccharomyces cerevisiae Strains Resulted in Enhanced Production of 2-Phenylethanol by Biotransformation of L-Phenylalanine

2017 ◽  
Vol 27 (2) ◽  
pp. 81-90 ◽  
Author(s):  
Jolanta Mierzejewska ◽  
Aleksandra Tymoszewska ◽  
Karolina Chreptowicz ◽  
Kamil Krol
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Batch Culture ◽  
Fold Increase ◽  
Biotechnological Production ◽  
Aromatic Alcohol ◽  
Enhanced Production ◽  
Yeast Strains ◽  
First Time

2-Phenylethanol (2-PE) is an aromatic alcohol with a rosy scent which is widely used in the food, fragrance, and cosmetic industries. Promising sources of natural 2-PE are microorganisms, especially yeasts, which can produce 2-PE by biosynthesis and biotransformation. Thus, the first challenging goal in the development of biotechnological production of 2-PE is searching for highly productive yeast strains. In the present work, 5 laboratory <i>Saccharomyces cerevisiae</i> strains were tested for the production of 2-PE. Thereafter, 2 of them were hybridized by a mating procedure and, as a result, a new diploid, <i>S. cerevisiae</i> AM1-d, was selected. Within the 72-h batch culture in a medium containing 5 g/L of <smlcap>L</smlcap>-phenylalanine, AM1-d produced 3.83 g/L of 2-PE in a shaking flask. In this way, we managed to select the diploid <i>S. cerevisiae</i> AM1-d strain, showing a 3- and 5-fold increase in 2-PE production in comparison to parental strains. Remarkably, the enhanced production of 2-PE by the hybrid of 2 yeast laboratory strains is demonstrated here for the first time.

scholarly journals Deciphering the Mechanism of Glyphosate Resistance in Amaranthus palmeri by Cytogenomics

2022 ◽  
pp. 1-7
Author(s):  
Dal-Hoe Koo ◽  
Rajendran Sathishraj ◽  
Bernd Friebe ◽  
Bikram S. Gill
Keyword(s):  
Genome Size ◽  
Copy Number ◽  
Fold Increase ◽  
Glyphosate Resistance ◽  
Resistance Mechanisms ◽  
Fish Analysis ◽  
Amaranthus Palmeri ◽  
Artificial Chromosomes ◽  
Genetic Elements ◽  
Meiotic Chromosomes

In agriculture, various chemicals are used to control the weeds. Out of which, glyphosate is an important herbicide invariably used in the cultivation of glyphosate-resistant crops to control weeds. Overuse of glyphosate results in the evolution of glyphosate-resistant weeds. Evolution of glyphosate resistance (GR) in <i>Amaranthus palmeri</i> (AP) is a serious concern in the USA. Investigation of the mechanism of GR in AP identified different resistance mechanisms of which <i>5-enolpyruvylshikimate-3-phosphate synthase</i> (<i>EPSPS</i>) gene amplification is predominant. Molecular analysis of GR AP identified the presence of a 5- to &#x3e;160-fold increase in copies of the <i>EPSPS</i> gene than in a glyphosate-susceptible (GS) population. This increased copy number of the <i>EPSPS</i> gene increased the genome size ranging from 3.5 to 11.8%, depending on the copy number compared to the genome size of GS AP. FISH analysis using a 399-kb <i>EPSPS</i> cassette derived from bacterial artificial chromosomes (BACs) as probes identified that amplified <i>EPSPS</i> copies in GR AP exist in extrachromosomal circular DNA (eccDNA) in addition to the native copy in the chromosome. The <i>EPSPS</i> gene-containing eccDNA having a size of ∼400 kb is termed <i>EPSPS</i>-eccDNA and showed somatic mosacism in size and copy number. <i>EPSPS</i>-eccDNA has a genetic mechanism to tether randomly to mitotic or meiotic chromosomes during cell division or gamete formation and is inherited to daughter cells or progeny generating copy number variation. These eccDNAs are stable genetic elements that can replicate and exist independently. The genomic characterization of the <i>EPSPS</i> locus, along with the flanking regions, identified the presence of a complex array of repeats and mobile genetic elements. The cytogenomics approach in understanding the biology of <i>EPSPS</i>-eccDNA sheds light on various characteristics of <i>EPSPS</i>-eccDNA that favor GR in AP.

Evidence That Selected Amplification of a Bacterial lac Frameshift Allele Stimulates Lac+ Reversion (Adaptive Mutation) With or Without General Hypermutability

Genetics ◽  
2002 ◽  
Vol 161 (3) ◽  
pp. 945-956 ◽  
Author(s):  
E Susan Slechta ◽  
Jing Liu ◽  
Dan I Andersson ◽  
John R Roth
Keyword(s):  
Model Selection ◽  
Direct Effect ◽  
Mutation Rate ◽  
Side Effect ◽  
Copy Number ◽  
Fold Increase ◽  
Genetic System ◽  
Adaptive Mutation ◽  
E Coli ◽  
Frameshift Mutant

Abstract In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac+ revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification—induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.

scholarly journals Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S-adenosylmethionine by coupling of an engineered S-adenosylmethionine pathway and the tricarboxylic acid cycle

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Liying Ruan ◽  
Lu Li ◽  
Dian Zou ◽  
Cong Jiang ◽  
Zhiyou Wen ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Bacillus Amyloliquefaciens ◽  
Tricarboxylic Acid Cycle ◽  
Fold Increase ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Scale Production ◽  
Free Medium ◽  
Enhanced Production ◽  
The Tca Cycle

Abstract Background S-Adenosylmethionine (SAM) is a critical cofactor involved in many biochemical reactions. However, the low fermentation titer of SAM in methionine-free medium hampers commercial-scale production. The SAM synthesis pathway is specially related to the tricarboxylic acid (TCA) cycle in Bacillus amyloliquefaciens. Therefore, the SAM synthesis pathway was engineered and coupled with the TCA cycle in B. amyloliquefaciens to improve SAM production in methionine-free medium. Results Four genes were found to significantly affect SAM production, including SAM2 from Saccharomyces cerevisiae, metA and metB from Escherichia coli, and native mccA. These four genes were combined to engineer the SAM pathway, resulting in a 1.42-fold increase in SAM titer using recombinant strain HSAM1. The engineered SAM pathway was subsequently coupled with the TCA cycle through deletion of succinyl-CoA synthetase gene sucC, and the resulted HSAM2 mutant produced a maximum SAM titer of 107.47 mg/L, representing a 0.59-fold increase over HSAM1. Expression of SAM2 in this strain via a recombinant plasmid resulted in strain HSAM3 that produced 648.99 mg/L SAM following semi-continuous flask batch fermentation, a much higher yield than previously reported for methionine-free medium. Conclusions This study reports an efficient strategy for improving SAM production that can also be applied for generation of SAM cofactors supporting group transfer reactions, which could benefit metabolic engineering, chemical biology and synthetic biology.

scholarly journals Molecular biological characterization of an azole-resistant Candida glabrata isolate.

1997 ◽  
Vol 41 (10) ◽  
pp. 2229-2237 ◽  
Author(s):  
P Marichal ◽  
H Vanden Bossche ◽  
F C Odds ◽  
G Nobels ◽  
D W Warnock ◽  
...  
Keyword(s):  
Copy Number ◽  
Candida Glabrata ◽  
Fold Increase ◽  
Resistant Isolate ◽  
Protein Patterns ◽  
Gene Encoding ◽  
Two Dimensional Gel Electrophoresis ◽  
Azole Antifungals ◽  
Susceptible Isolate ◽  
Lanosterol Demethylase

Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14alpha-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14alpha-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demonstrated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in fluconazole-free medium and correlated with reversion to susceptibility. The greater abundance of the amplified chromosome induced pronounced differences in the protein patterns of the susceptible and revertant isolates versus that of the resistant isolate, as demonstrated by two-dimensional gel electrophoresis (2D-GE). Densitometry of the 2D-GE product indicated upregulation of at least 25 proteins and downregulation of at least 76 proteins in the resistant isolate.

scholarly journals Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization

2007 ◽  
Vol 75 (10) ◽  
pp. 4804-4816 ◽  
Author(s):  
Yongkai Mo ◽  
Natalie M. Quanquin ◽  
William H. Vecino ◽  
Uma Devi Ranganathan ◽  
Lydia Tesfa ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Mycobacterium Smegmatis ◽  
Copy Number ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
J774 Cells

ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

scholarly journals Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

1999 ◽  
Vol 337 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Junlong ZHANG ◽  
Christopher D. BYRNE
Keyword(s):  
Reverse Transcriptase ◽  
Copy Number ◽  
Accurate Determination ◽  
Fold Increase ◽  
Pcr Primers ◽  
Cdna Synthesis ◽  
Reverse Transcriptase Pcr ◽  
Target Mrna ◽  
Mrna Copy Number ◽  
Synthesis Reactions

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a ≈ 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P< 0.001, n = 4), and a ≈ 4-fold increase in copy number with a specific hexamer (P< 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

scholarly journals Screening and Evaluation of a Potential Bacteriocin Producing Lactobacillus in Milk and Dairy Products Collected from Puducherry

2021 ◽  
Vol 14 (4) ◽  
pp. 1501-1507
Author(s):  
T Punithavalli
Keyword(s):  
16S Rrna ◽  
Dairy Products ◽  
Fold Increase ◽  
Bacteriocin Production ◽  
World Health ◽  
Partial Sequence ◽  
Lactobacillus Species ◽  
Screening Process ◽  
Enhanced Production ◽  
Milk And Dairy Products

In an array of identifying safe antimicrobial compounds, bacteriocin producing Lactobacillus strain have been investigated in this study from the daily consuming food resources of humans. Till now, the best studied bacteriocins are nisin A produced by Lactobacillus lactis and pedocin PA-1 synthesized by Pediococcus acidilactici which have been accredited as a preservative in the food industries by the World Health Organization (WHO). For this study, four different milk and dairy products viz., curd, cheese, yoghurt and butter were collected from the local markets of Karaikal region, Puducherry, India and were used for the isolation of Lactobacillus species using MRS agar. Totally, five morphologically distinct strains were collected and were initially named as MPD 1 to MPD 5. During the screening process of bacteriocin production, the strain MPD 5 showed maximum antimicrobial activity against Vibrio cholerae MTCC 3906 with 900AU/ml. This strain was molecular identified as Lactiplantibacillus plantarum MDP 5 based on 16S rRNA partial sequence method. This 16S rRNA partial sequence was submitted to the NCBI nucleotide GenBank and provided with the accession number, MW301154.1. Further, this strain revealed an enhanced production of bacteriocin using the standardized physicochemical factors such as pH 7, 35°C, 2% fructose and 1% peptone. Furthermore, these optimal conditions revealed more than 2-fold increase in the bacteriocin production. All the above information suggesting the possibilities of bacteriocin for the bioindustrial production using the L. plantarum MDP 5 of this study and its future prospects for the investigation of biocidal activities against many highly infectious pathogens of human and veterinary.

scholarly journals JAX-CNV: A whole genome sequencing-based algorithm for copy number detection at clinical grade level

2021 ◽  
Author(s):  
Wan-Ping Lee ◽  
Qihui Zhu ◽  
Xiaofei Yang ◽  
Silvia Liu ◽  
Eliza Cerveira ◽  
...  
Keyword(s):  
False Discovery Rate ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Copy Number Variant ◽  
Fold Increase ◽  
Chromosomal Microarray ◽  
Whole Genome ◽  
False Discovery ◽  
Calling Algorithm

We aimed to develop a whole genome sequencing (WGS)-based copy number variant (CNV) calling algorithm with the potential of replacing chromosomal microarray assay (CMA) for clinical diagnosis. JAX-CNV is thus developed for CNV detection from WGS. The performance of this CNV calling algorithm was evaluated in a blinded manner on 31 samples and compared to the results of clinically-validated CMAs. Comparing to 112 CNVs reported by clinically-validated CMAs of the 31 samples, JAX-CNV is 100% recalling them. Besides, JAX-CNV identified an average of 30 CNVs per individual that is an approximately seven-fold increase compared to calls of clinically-validated CMAs. Experimental validation of 24 randomly selected CNVs, showed one false positive (i.e., a false discovery rate of 4.17%). A robustness test on lower-coverage data revealed a 100% sensitivity for CNVs greater than 300 kb (the current threshold for College of American Pathologists) down to 10x coverage. For CNVs greater than 50 kb, sensitivities were 100% for coverages deeper than 20x, 97% for 15x, and 95% for 10x. We developed a WGS-based CNV pipeline, including this newly developed CNV caller JAX-CNV, and found it capable of detecting CMA reported CNVs at 100% sensitivity with about 4% false discovery rate. We propose that JAX-CNV could be further examined in a multi-institutional study to justify the transition of first-tier genetic testing from CMAs to WGS. JAX-CNV is available on https://github.com/TheJacksonLaboratory/JAX-CNV.

scholarly journals Enhanced Production of Pediocin PA-1 and Coproduction of Nisin and Pediocin PA-1 by Lactococcus lactis

1999 ◽  
Vol 65 (10) ◽  
pp. 4443-4450 ◽  
Author(s):  
Nikki Horn ◽  
María I. Martínez ◽  
José M. Martínez ◽  
Pablo E. Hernández ◽  
Michael J. Gasson ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Copy Number ◽  
Antimicrobial Agents ◽  
Expression System ◽  
Pediococcus Acidilactici ◽  
Genetic Determinants ◽  
Heterologous Expression System ◽  
Food Grade ◽  
Enhanced Production ◽  
Mature Part

ABSTRACT The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides.Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martı́nez, J. M. Martı́nez, P. E. Hernández, M. J. Gasson, J. M. Rodrı́guez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818–823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain,Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.

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