No.

Contact Our BIO-INNOVATION CENTER

Technology to Boost Production Yields- With a Focus on tRNA Availability NAGASE & CO. developed a technology that uses high-availability transfer RNA(tRNA) to improve production yields in microbial fermentation.
Summary
Technologies for mass-producing proteins in microorganisms are essential issues for not only to academic
research but also industrial production (Figure 1). As one of the solution to the problems, a host cell istransfected with a gene which may contain portions that code for tRNA corresponding to rare codons (i.e.,codons infrequently used by the host cell). Also, the rare codons in the gene are replaced with frequently
used synonymous codons to generate a codon-optimized gene 1-3.
The frequency of tRNA used in the host cell is defined as the availability rate (Figure 2). Transfecting hostcells with genes that code for highly available tRNA improve the yield of the target compound produced by thehost cell 4.

Higher yields

Microorganism cells (production plant)

Proteins

Target gene

ChemicalstRNA

Figure 1 Producing substances with microbial fermentation

Description
1. What does tRNA do, and what does
“availability rate” mean?
In the protein translation process within cells, atRNA carries an amino acid that corresponds to agiven codon. Different types of tRNA, however, areused at varying frequencies during translation.
The concept of tRNA availability is key to thetechnology discussed here. The availability rate oftRNA corresponding to the codon AAA, forexample, represents the ratio of tRNA(corresponding to the codon AAA) gene number tothe frequency of codon AAA appears in all coded
genes in the genome.
Figure 2 shows the availability rates of tRNA in hostmicroorganisms commonly used in industrial
production. Importantly, high-availability tRNAdiffers from species to species.

TAC
TAC
TGG
TGG
ACT
ACT
ACG
ACG

GTT
GTC GTGGTG GTTGTA GTC
GTA
TAT
TAT

GCA
GCA

GCC GCGGCC GCG GCTGCT AGAAGAAGG
AGG
CGA
CGA
CGC
CGC
CGG
CGG
CGT
CGT

ACC
ACC
ACA
ACA
TCT
TCT

AAC
AAC
AAT
AAT

GAC
GAC
GAT
GAT

TCG
TCG
TCC
TCC

TGC
TGC
TGT
TGT

TCA
TCA
AGT
AGT

CAA
CAA
CAG
CAG
GAA
GAA

AGC
AGC
CCT
CCT
CCG
CCG
CCC
CCC

GAG
GAG
GGA
GGA

CCA
CCA
TTT
TTT
TTC
TTC
ATGATG
AAG
AAG
AAA
AAA TTG
TTG TTATTA CTTCTT CTG
CTG

GGC
GGC
GGG
GGG
GGT
GGT
CAC
CAC
CAT
CAT
ATA
ATC ATAATT ATC
CTA ATTCTC
CTC CTA

Streptomyces coelicolor A3(2)Escherichia coli K12
Corynebacterium glutamicum
Bacillus subtilis
Schizosaccharomyces pombe
Aspergillus fumigatus

Figure 2 Distribution of tRNA availability

Streptomyces coeli
coeli
Streptomyces

Escherichia coli
coli K12
K12
Escherichia
Corynebacterium
Corynebacterium gg

Bacillus subtilus
subtilus
Bacillus
Schizosaccharomyc
Schizosaccharomyc

Aspergillus fumigatfumigatAspergillus

No.

2. What are the effects of using high-availability tRNA?
Protein yields in the expression host Streptomyces violaceoruber 1326 are shown in Figure 3. The yield of
more than half of the proteins increased significantly when the host was transfected with a gene coding fortwo high-availability tRNA types as determined through theoretical calculations. Furthermore, similar
increases in production yields were obtained in the expression host Escherichia coli K12.
Transfecting Streptomyces hosts with genes using high-availability tRNA also improved yields of non-protein
chemicals.
3.036.3expression in hostRelative
元株に対する発現量比

Boldface: p<0.05

2.5

2.3
2.021

11.2

1.8

1.0

1.6
1.5
1.1 1.1

1.6
1.4
1.2

1.3
0.90Proteins
Promoter

SSMP

PLD

SAV

xylFigure 3 Evaluation of protein yields

The Wide-ranging Effects of this Research
The concept of tRNA availability defined here is applicable to all organisms, microbial and otherwise, andtherefore has applications in many expression hosts now in use.

References
1. Rosenberg AH. et al. Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatilecodon test system. J.Bacteriol (1993) 175(3): 716-722
2. Brinkmann U. et al. High-level expression of recombinant genes in Escherichia coli is dependent on the availability of thednaY gene product. Gene (1989) 85(1): 109-114
3. Seidel HM et al. Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from
Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli. Biochemistry (1992) 31(9): 25982608
4. Method for improving yield of substance from micro-organism, and kit using said method. Japanese Patent, No. 5927593(2016)The NAGASE BIO-INNOVATION CENTER is committed to developing processes for efficiently
producing a wide range of compounds with proprietary fermentation technologies.

Contact Our BIO-INNOVATION CENTER© 2018-2022 NAGASE & CO., LTD.