Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Xue, Feiqun; Liu, Zihui; Yu, Yue; Wu, Yangjie; Jin, Yuxin; Yang, Mo; Ma, Lanqing

doi:10.3389/fbioe.2020.610506

Cited by 5 publications

(2 citation statements)

References 36 publications

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“…The codon optimization method generally replaced the rare codons in the target gene with the commonly used codon in the host cell without modifying the target protein [31]. Codon usage is critical to achieving adequate protein expression levels in E. coli [32], [33]. This technique can improve the production of various recombinant proteins expressed by E. coli [34], [35], [36].…”

Section: Resultsmentioning

confidence: 99%

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Rodiansyah¹,

Tan²,

Nugrahapraja³

2023

Int. J. Adv. Sci. Eng. Inf. Technol.

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Staphylococcal enterotoxin B (SEB) from Staphylococcus aureus is a potential therapeutic agent for cancer. SEB can activate the immune response, which could induce apoptosis of various cancer cells. This study aimed to design and produce a recombinant SEB protein using Escherichia coli BL21(DE3) expression system. We designed and optimized the codon of the inserted gene for E. coli and then transformed it into E. coli BL21(DE3). The transformants were verified by selective media containing kanamycin followed by colony PCR with T7 promoter and T7 terminator primers and DNA sequencing. The results showed that the SEBsyn encoding gene could be synthesized and cloned into pET-28a(+) expression plasmid. This recombinant plasmid that carried the SEB encoding gene (pET-28a_SEBsyn) was successfully transformed into E. coli BL21(DE3). The amplicons of colony PCR visualized in 2% agarose gel showed that transformants carrying the recombinant plasmid had an inserted gene length of about 1 kb. Gene inserts sequence verification by DNA sequencing resulted in 1,148 bp sequence consensus. The blastx analysis showed that it had the best hit with enterotoxin B in the NCBI database (accession id CAC6284025.1). The recombinant SEB protein was also successfully overexpressed in E. coli BL21(DE3) with 0.1 mM IPTG. Our recombinant SEB protein was dominantly insoluble in the inclusion body; however, some SEB protein was expressed as soluble. The molecular weight of the target protein is about 29 kDa.

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Section: Resultsmentioning

confidence: 99%

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Rodiansyah¹,

Tan²,

Nugrahapraja³

2023

Int. J. Adv. Sci. Eng. Inf. Technol.

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“…β-Carotene, β-cryptoxanthin, torulene, and torularhodin can be produced by R. glutinis (8). And also, R. glutinis can produce some important industrial enzymes, such as phenylalanine ammonia lyase (PAL), a key enzyme in the phenylalanine pathway for the removal of ammonia from phenylalanine to produce trans-cinnamic acid (9). In addition, R. glutinis can achieve the purpose of improving the soil environment by adsorbing heavy metal elements (10).…”

Section: Introductionmentioning

confidence: 99%

Carotenoid Biosynthesis: Genome-Wide Profiling, Pathway Identification in Rhodotorula glutinis X-20, and High-Level Production

Guo

et al. 2022

Front. Nutr.

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Rhodotorula glutinis, as a member of the family Sporidiobolaceae, is of great value in the field of biotechnology. However, the evolutionary relationship of R. glutinis X-20 with Rhodosporidiobolus, Sporobolomyces, and Rhodotorula are not well understood, and its metabolic pathways such as carotenoid biosynthesis are not well resolved. Here, genome sequencing and comparative genome techniques were employed to improve the understanding of R. glutinis X-20. Phytoene desaturase (crtI) and 15-cis-phytoene synthase/lycopene beta-cyclase (crtYB), key enzymes in carotenoid pathway from R. glutinis X-20 were more efficiently expressed in S. cerevisiae INVSc1 than in S. cerevisiae CEN.PK2-1C. High yielding engineered strains were obtained by using synthetic biology technology constructing carotenoid pathway in S. cerevisiae and optimizing the precursor supply after fed-batch fermentation with palmitic acid supplementation. Genome sequencing analysis and metabolite identification has enhanced the understanding of evolutionary relationships and metabolic pathways in R. glutinis X-20, while heterologous construction of carotenoid pathway has facilitated its industrial application.

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Integrated Experimental and Mathematical Modeling to Guide Microbial Biocatalysis: Pseudomonas putida Conversion of L‐Phenylalanine to trans‐Cinnamic Acid

Antimanon,

Jensen,

Woodley

2024

Biotech & Bioengineering

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ABSTRACTtrans‐Cinnamic acid (tCA) can be produced by the deamination of L‐phenylalanine using phenylalanine ammonia‐lyase (PAL). In bioprocesses, optimizing production processes to improve key performance metrics such as titer, rate, and yield has proved challenging. This study investigates tCA production in Pseudomonas putida KT2440 using a whole‐cell biocatalyst expressing four different PAL genes. Among these, RmXAL showed the highest titer and rate. Biocatalysis at pH 8.5 and 37°C were identified as the best conditions, giving a tCA concentration of 2.65 g L−1 and a production rate of 0.44 g L−1 h−1. To improve the metrics further, a decoupled bioprocess with various biocatalyst concentrations in the second stage was examined. With a whole‐cell biocatalyst concentration of 30 gDCW L−1, optimal process parameters were achieved, giving a titer of 29.88 g L−1, rate of 5.99 g L−1 h−1, yield on glucose of 0.27 g tCA g glucose−1, and yield on L‐phe of 0.75 g tCA g L‐phe−1. This study is the first report of a model that enables performance metrics to evaluate a suitable process for tCA production and provides valuable insights into tCA production using a decoupled bioprocess. This would appear applicable to larger‐scale production, paving the way for an efficient and sustainable industrial process.

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Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Cited by 5 publications

References 36 publications

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Expression of a Synthetic Staphylococcal Enterotoxin B in Escherichia coli BL21(DE3) for Cancer Therapy Development

Carotenoid Biosynthesis: Genome-Wide Profiling, Pathway Identification in Rhodotorula glutinis X-20, and High-Level Production

Integrated Experimental and Mathematical Modeling to Guide Microbial Biocatalysis: Pseudomonas putida Conversion of L‐Phenylalanine to trans‐Cinnamic Acid

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