Engineering the Translational Machinery for Biotechnology Applications

Wang, Tianwen; Liang, Chen; An, Yafei; Xiao, Sa; Xu, Hongjv; Zheng, Minying; Liu, Lu; Wang, Gaozhan; Nie, Lei

doi:10.1007/s12033-020-00246-y

Cited by 8 publications

(7 citation statements)

References 77 publications

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“…A major limiting factor for the practical utilization of enzymes containing ncAAs is their low expression yields . Fortunately, recent breakthroughs successfully increased the protein expression levels and enabled the multisite incorporation of ncAA by engineering ribosomes or deleting release factors. ,,, The primary point of consideration for creating novel enzymes or enhancing an enzyme’s functionality by expanding the amino acid alphabet should be its cost-effectiveness. The high cost of the ncAAs is a significant bottleneck for large-scale applications of enzymes containing ncAAs .…”

Section: Challenges and Prospectsmentioning

confidence: 99%

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

et al. 2021

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The two main strategies for enzyme engineering, directed evolution and rational design, have found widespread applications in improving the intrinsic activities of proteins. Although numerous advances have been achieved using these ground-breaking methods, the limited chemical diversity of the biopolymers, restricted to the 20 canonical amino acids, hampers creation of novel enzymes that Nature has never made thus far. To address this, much research has been devoted to expanding the protein sequence space via chemical modifications and/or incorporation of noncanonical amino acids (ncAAs). This review provides a balanced discussion and critical evaluation of the applications, recent advances, and technical breakthroughs in biocatalysis for three approaches: (i) chemical modification of cAAs, (ii) incorporation of ncAAs, and (iii) chemical modification of incorporated ncAAs. Furthermore, the applications of these approaches and the result on the functional properties and mechanistic study of the enzymes are extensively reviewed. We also discuss the design of artificial enzymes and directed evolution strategies for enzymes with ncAAs incorporated. Finally, we discuss the current challenges and future perspectives for biocatalysis using the expanded amino acid alphabet.

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Section: Challenges and Prospectsmentioning

confidence: 99%

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

et al. 2021

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“…Directly manipulating tRNAs in the cell is unrealistic, but it is convenient in CFPS without a membrane barrier, freeing a large number of sense codons for ncAA incorporation by tRNA manipulation ( Tajima et al, 2018 ). Considering the one-to-one correspondence between tRNAs and codons, artificial codon tables, and even minimum codon combinations can be achieved by tRNA manipulation in CFPS ( Calles et al, 2019 ; Wang et al, 2020 ). Recently, several tRNA level methods were developed, including tRNA pool replacement, elimination using antisense oligonucleotides, and tRNA-specific enzymatic degradation ( Figure 2C ).…”

Section: Strategies For Eliminating Competitionmentioning

confidence: 99%

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

Wang

Qiao

et al. 2020

Front. Bioeng. Biotechnol.

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Cell-free protein synthesis (CFPS) has emerged as a novel protein expression platform. Especially the incorporation of non-canonical amino acids (ncAAs) has led to the development of numerous flexible methods for efficient and extensive expression of artificial proteins. Approaches were developed to eliminate the endogenous competition for ncAAs and engineer translation factors, which significantly enhanced the incorporation efficiency. Furthermore, in vitro aminoacylation methods can be conveniently combined with cell-free systems, extensively expanding the available ncAAs with novel and unique moieties. In this review, we summarize the recent progresses on the efficient and extensive incorporation of ncAAs by different strategies based on the elimination of competition by endogenous factors, translation factors engineering and extensive incorporation of novel ncAAs coupled with in vitro aminoacylation methods in CFPS. We also aim to offer new ideas to researchers working on ncAA incorporation techniques in CFPS and applications in various emerging fields.

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“…Directly manipulating tRNAs in the cell is unrealistic, but it is convenient in CFPS without a membrane barrier, freeing a large number of sense codons for ncAA incorporation by tRNA manipulation [62]. Considering the oneto-one correspondence between tRNAs and codons, artificial codon tables, and even minimum codon combinations can be achieved by tRNA manipulation in CFPS [63,64]. Recently, several tRNA level methods were developed, including tRNA pool replacement, elimination using antisense oligonucleotides, and tRNA-specific enzymatic degradation (Figure 2C).…”

Section: Manipulation Of Trnasmentioning

confidence: 99%

Emerging Methods for Efficient and Extensive Incorporation of Non Canonical Amino Acids using Cell-free Systems

Wu¹,

Wang²,

Qiao³

et al. 2020

Prime Archives in Biotechnology

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Engineering the Translational Machinery for Biotechnology Applications

Cited by 8 publications

References 77 publications

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

Emerging Methods for Efficient and Extensive Incorporation of Non Canonical Amino Acids using Cell-free Systems

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