Metabolic engineering of capsular polysaccharides

2018 ◽  
Vol 2 (3) ◽  
pp. 337-348 ◽  
Author(s):  
Asher Williams ◽  
Robert J. Linhardt ◽  
Mattheos A.G. Koffas
Keyword(s):  
Metabolic Engineering ◽  
De Novo ◽  
Gene Knockout ◽  
Polysialic Acid ◽  
Genetically Engineered ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Level Control ◽  
Biotechnological Processes ◽  
Type Strains

With rising concerns about sustainable practices, environmental complications, and declining resources, metabolic engineers are transforming microorganisms into cellular factories for producing capsular polysaccharides (CPSs). This review provides an overview of strategies employed for the metabolic engineering of heparosan, chondroitin, hyaluronan, and polysialic acid — four CPSs that are of interest for manufacturing a variety of biomedical applications. Methods described include the exploitation of wild-type and engineered native CPS producers, as well as genetically engineered heterologous hosts developed through the improvement of naturally existing pathways or newly (de novo) designed ones. The implementation of methodologies like gene knockout, promoter engineering, and gene expression level control has resulted in multiple-fold improvements in CPS fermentation titers compared with wild-type strains, and substantial increases in productivity, reaching as high as 100% in some cases. Optimization of these biotechnological processes can permit the adoption of industrially competitive engineered microorganisms to replace traditional sources that are generally toxic, unreliable, and inconsistent in product quality.

scholarly journals RAD51-Dependent Break-Induced Replication in Yeast

2004 ◽  
Vol 24 (6) ◽  
pp. 2344-2351 ◽  
Author(s):  
Allison P. Davis ◽  
Lorraine S. Symington
Keyword(s):  
High Frequency ◽  
De Novo ◽  
Recombinational Repair ◽  
Wild Type ◽  
Chromosome Fragment ◽  
Chromosome Fragmentation ◽  
Strand Invasion ◽  
Chromosomal Sequences ◽  
Break Induced Replication ◽  
Type Strains

ABSTRACT A chromosome fragmentation assay was used to measure the efficiency and genetic control of break-induced replication (BIR) in Saccharomyces cerevisiae. Formation of a chromosome fragment by de novo telomere generation at one end of the linear vector and recombination-dependent replication of 100 kb of chromosomal sequences at the other end of the vector occurred at high frequency in wild-type strains. RAD51 was required for more than 95% of BIR events involving a single-end invasion and was essential when two BIR events were required for generation of a chromosome fragment. The similar genetic requirements for BIR and gene conversion suggest a common strand invasion intermediate in these two recombinational repair processes. Mutation of RAD50 or RAD59 conferred no significant defect in BIR in either RAD51 or rad51 strains. RAD52 was shown to be essential for BIR at unique chromosomal sequences, although rare recombination events were detected between the subtelomeric Y′ repeats.

scholarly journals Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

2003 ◽  
Vol 69 (2) ◽  
pp. 812-819 ◽  
Author(s):  
Jung-Hoon Bae ◽  
Jung-Hoon Sohn ◽  
Chang-Seo Park ◽  
Joon-Shick Rhee ◽  
Eui-Sung Choi
Keyword(s):  
Metabolic Engineering ◽  
De Novo ◽  
Integration Site ◽  
Fold Increase ◽  
Selection Marker ◽  
Serine Palmitoyltransferase ◽  
Transformation System ◽  
Wild Type ◽  
Gene Encoding ◽  
Integrative Transformation

ABSTRACT We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.

scholarly journals Enhanced pyruvate metabolism in plastids by overexpression of plastidial pyruvate transporter in Phaeodactylum tricornutum

2020 ◽  
Author(s):  
Seungbeom Seo ◽  
Joon Kim ◽  
Jun-Woo Lee ◽  
Onyou Nam ◽  
Kwang Suk Chang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Phaeodactylum Tricornutum ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Biofuel Production ◽  
Wild Type ◽  
Strain Development ◽  
Acid Biosynthesis ◽  
Pyruvate Metabolism ◽  
Lipid Contents

Abstract BackgroundThe development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development.ResultsWe identified pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation.ConclusionTo regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the plastidial pyruvate transporter in P. tricornutum. They showed that compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids.

scholarly journals Self-Limiting OX513A Aedes aegypti Demonstrate Full Susceptibility to Currently Used Insecticidal Chemistries as Compared to Indian Wild-Type Aedes aegypti

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Prabhakargouda B. Patil ◽  
Kevin J. Gorman ◽  
Shaibal K. Dasgupta ◽  
K. V. Seshu Reddy ◽  
Shirish R. Barwale ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Mortality Rates ◽  
Standard Test ◽  
Genetically Engineered ◽  
World Health ◽  
Wild Type ◽  
Test Procedures ◽  
Male And Female ◽  
Health Organization ◽  
Type Strains

OX513A Aedes aegypti is a genetically engineered strain carrying a self-limiting gene. Studies in several countries have shown the effectiveness of the strain at reducing pest Aedes aegypti populations. As a component of biosafety assessments relevant to Indian environments, OX513A and two Indian wild-type Ae. aegypti strains (from Aurangabad and Delhi) were tested for susceptibility to a range of commonly used insecticides in India, such as dichlorodiphenyltrichloroethane (DDT), malathion, deltamethrin, and permethrin using World Health Organization (WHO) testing kits and following WHO standard test procedures. Knockdown times (KDT) for all compounds were determined separately for male and female adults of the three mosquito strains. Results indicated that adults of OX513A, Aurangabad, and Delhi strains were resistant to DDT, yielding mortality rates of 90.9, 87.4, and 44.4% and 70.1, 3.0, and 6.0% for male and female adults, respectively. In contrast, adults of all three strains were found to be susceptible to malathion, deltamethrin, and permethrin, exhibiting mortalities between 98 and 100%. The magnitudes of susceptibility, based on the KDT50 values, were greater in the OX513A strain, as compared to wild-type strains of Ae. aegypti for all insecticides tested. The results confirm that, aside from historical resistance to DDT, OX513A has retained full sensitivity to these commonly used compounds and exhibits responses akin to those of susceptible Indian wild-type strains.

scholarly journals Functional Characterization of Transporters for L-Aspartate in Bacillus licheniformis

2021 ◽  
Author(s):  
Hanrong Wang ◽  
Youran Li ◽  
Fengxu Xiao ◽  
Yupeng Zhang ◽  
Guiyang Shi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Gene Knockout ◽  
Single Gene ◽  
Functional Characterization ◽  
Transport Systems ◽  
Amino Acid Transporters ◽  
Wild Type ◽  
Beta Alanine ◽  
Type Strains

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms’ cell growth and metabolism. However, although biochemically characterized, most amino acid transporters remain unknown at the molecular level in Bacillus licheniformis. This study focuses on the molecular and functional characterizations of three transporters, YdgF, YvbW, and YveA, mainly when catalyzing the cross-membrane flux of L-Aspartate. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains can alleviate the inhibition to some extent. Upon overexpression, the recombinant proteins mediate the accumulation of L-aspartate to varying degrees. Compared with wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this paper focuses on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine.

scholarly journals Enhanced pyruvate metabolism in plastids by overexpression of putative plastidial pyruvate transporter in Phaeodactylum tricornutum

2020 ◽  
Author(s):  
Seungbeom Seo ◽  
Joon Kim ◽  
Jun-Woo Lee ◽  
Onyou Nam ◽  
Kwang Suk Chang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Phaeodactylum Tricornutum ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Biofuel Production ◽  
Wild Type ◽  
Strain Development ◽  
Acid Biosynthesis ◽  
Pyruvate Metabolism ◽  
Lipid Contents

Abstract Background: The development of microalgal strains for enhanced biomass and biofuel production has received increased attention. Moreover, strain development via metabolic engineering for commercial production is being considered as the most efficient strategy. Pyruvate is an essential metabolite in the cells and plays an essential role in amino acid biosynthesis and de novo fatty acid biosynthesis in plastids. Although pyruvate can be a valuable target for metabolic engineering, its transporters have rarely been studied in microalgae. In this study, we aimed to identify the plastidial pyruvate transporter of Phaeodactylum tricornutum and utilize it for strain development.Results: We identified putative pyruvate transporter localized in the plastid membrane of Phaeodactylum tricornutum. Transformants overexpressing the pyruvate transporter were generated to increase the influx of pyruvate into plastids. Overexpression of a plastidial pyruvate transporter in P. tricornutum resulted in enhanced biomass (13.6% to 21.9%), lipid contents (11% to 30%), and growth (3.3% to 8.0%) compared to those of wild type during one-stage cultivation. Conclusion: To regulate the pyruvate influx and its metabolism in plastids, we generated transformants overexpressing the putative plastidial pyruvate transporter in P. tricornutum. They showed that its overexpression for compartmentalizing pyruvate in plastids could be an attractive strategy for the effective production of biomass and lipids with better growth, via enhanced pyruvate metabolism in plastids.

scholarly journals Comparative Transcriptomics and Gene Knockout Reveal Virulence Factors of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis

2021 ◽  
Vol 7 (12) ◽  
pp. 1001
Author(s):  
Xinmei Fang ◽  
Peng Yan ◽  
Mingmin Guan ◽  
Shan Han ◽  
Tianmin Qiao ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Gene Knockout ◽  
Disease Index ◽  
Economic Losses ◽  
Rna Seq ◽  
Wild Type ◽  
Relative Growth ◽  
Rice Tissue ◽  
Arthrinium Phaeospermum ◽  
Type Strains

Arthrinium phaeospermum can cause branch wilting of Bambusa pervariabilis × Dendrocalamopsis grandis, causing great economic losses and ecological damage. A. phaeospermum was sequenced in sterile deionized water (CK), rice tissue (T1) and B. pervariabilis × D. grandis (T2) fluid by RNA-Seq, and the function of Ctf1β 1 and Ctf1β 2 was verified by gene knockout. There were 424, 471 and 396 differentially expressed genes between the T2 and CK, T2 and T1, and CK and T1 groups, respectively. Thirty DEGs had verified the change in expression by fluorescent quantitative PCR. Twenty-nine DEGs were the same as the expression level in RNA-Seq. In addition, ΔApCtf1β 1 and ΔApCtf1β 2 showed weaker virulence by gene knockout, and the complementary strains Ctf1β 1 and Ctf1β 2 showed the same virulence as the wild-type strains. Relative growth inhibition of ΔApCtf1β 1 and ΔApCtf1β was significantly decreased by 21.4% and 19.2%, respectively, by adding H2O2 compared to the estimates from the wild-type strain and decreased by 25% and 19.4%, respectively, by adding Congo red. The disease index of B. pervariabilis × D. grandis infected by two mutants was significantly lower than that of wild type. This suggested that Ctf1β genes are required for the stress response and virulence of A. phaeospermum.

scholarly journals Functional Characterization of Transporters for L-Aspartate in Bacillus licheniformis

Fermentation ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 22
Author(s):  
Hanrong Wang ◽  
Youran Li ◽  
Fengxu Xiao ◽  
Yupeng Zhang ◽  
Guiyang Shi ◽  
...  
Keyword(s):  
Bacillus Licheniformis ◽  
Gene Knockout ◽  
Single Gene ◽  
Functional Characterization ◽  
Transport Systems ◽  
Wild Type ◽  
Carbon And Nitrogen ◽  
Influx Transport ◽  
Amino Acid Efflux ◽  
Type Strains

Amino acid efflux and influx transport systems play vital roles in industrial microorganisms’ cell growth and metabolism. However, although biochemically characterized, most of them remain unknown at the molecular level in Bacillus licheniformis. In this study, three proteins, namely, YdgF, YvbW, and YveA, were predicted to be involved in the active transport of L-aspartate (L-Asp). This was verified by manipulating their encoding genes. When growing in the minimal medium with L-Asp as the only carbon and nitrogen source, the growth of strains lacking proteins YdgF, YvbW, and YveA was significantly inhibited compared with the wild-type strains, while supplementing the expression of the corresponding proteins in the single-gene knockout strains could alleviate the inhibition. Upon overexpression, the recombinant proteins mediated the accumulation of L-aspartate to varying degrees. Compared with the wild-type strains, the single knockout strains of the three protein genes exhibited reduced absorption of L-aspartate. In addition, this study focused on the effects of these three proteins on the absorption of β-alanine, L-glutamate, D-serine, D-alanine, and glycine.

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