Utilization of rare codon-rich markers for screening amino acid overproducers

Abstract

The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation. Theoretically, disrupted or retarded translation caused by the lack of charged rare tRNAs can be partially restored by feeding or intracellular synthesis of the corresponding amino acids. Inspired by this assumption, we develop a screening or selection system for obtaining overproducers of a target amino acid by replacing its common codons with the corresponding synonymous rare alternative in the coding sequence of selected reporter proteins or antibiotic-resistant markers. Results show that integration of rare codons can inhibit gene translations in a frequency-dependent manner. As a proof-of-concept, Escherichia coli strains overproducing L-leucine, L-arginine or L-serine are successfully selected from random mutation libraries. The system is also applied to Corynebacterium glutamicum to screen out L-arginine overproducers. This strategy sheds new light on obtaining and understanding amino acid overproduction strains.

Fig. 1

Amino acid productions and codon usage. a Global productions of amino acids (left), the annual productions (right, represented by color intensity), and the fermentation titer (right, represented by bar height) for nine selected amino acids. b After taken up by the cells (i), the amino acid analogues (orange square) compete with the corresponding natural amino acids (blue hexagon) for the finite tRNAs, a step catalyzed by the aminoacyl-tRNA synthetase (aaRS). The analogues could be blocked (ii) or pumped outside of the cells (iii). c Codon usage and the fraction of tRNAs (bubble diameter) in E. coli. The fraction of individual tRNA out of the total tRNA was derived from E. coli W1485, a K strain derivative at a growth rate of 0.4 doublings h^–1. d For an exogenous gene, replacing its codons (e.g. leucine codon) with synonymous ones that are recognized by the most abundant tRNAs for a specific host would typically improve the expression of the desired protein (upper box). On the contrary, the rare tRNAs have lower chances to be charged with the corresponding amino acids, switching to the rare alternatives (e.g. leucine codon CTA for E. coli) that pair with the low-abundance tRNAs would dramatically slow down protein expression (lower box). Theoretically, the retarded protein expression should be restored by increased intracellular concentrations of the corresponding amino acids

Fig. 2

Effects of the frequency of leucine rare codon CTA on protein expressions. a Different numbers of the leucine codons on the wild-type kan^R were replaced by the rare one CTA, generating kan^R-RC6, kan^R-RC16, kan^R-RC26, and kan^R-RC29; the leucine codons on the wild-type gfp and ppg were also replaced by the rare alternative, generating gfp-RC and ppg-RC, respectively. b Influences of rare codon frequency on cell OD₆₀₀ for E. coli strains harboring the rare codon-rich kan^R (***P < 0.001 as determined by two-tailed t test). c Effects of the incorporation of leucine rare codon CTA and l-leucine feeding on GFP expression. d Effects of the incorporation of leucine rare codon CTA on PPG expression as indicated by the differences in color development. Values and error bars represent the mean and the s.d. (n = 3)

Fig. 3

Cell growth restored by feeding the corresponding l-amino acids. a Effects of feeding l-leucine or a mixture of three amino acids (3AA: l-leucine, l-valine and l-isoleucine) on the cell growth for E. coli strains harboring kan^R genes with 6−29 leucine rare codons (kan^R-RC6, kan^R-RC16, kan^R-RC26, kan^R-RC29). b Changes in cell ODs after feeding l-arginine to E. coli and C. glutamicum strains harboring kan^R genes in which eight arginine codons were replaced by its rare alternatives (encoded by pAKR-RC8 or pKan-CG-RC8), and the growth restoration after feeding l-serine to E. coli strains carrying spec^R which was rich in serine rare codon (encoded by pSSer-RC17). Values and error bars represent the mean and the s.d. (n = 3). **P < 0.01, ***P < 0.001 as determined by two-tailed t test

Fig. 4

Selection stringency mediated by copy number and promoter strength. The kan^R, kan^R-RC29, and kan^R-RC8 represent the wild-type kan^R, the kan^R containing leucine rare codon CTA, and the kan^R containing arginine rare codon AGG, respectively. a The effects of different ORIs on the cell OD₆₀₀ for strains harboring the wild-type and the rare codon-rich markers. b The effects of constitutive and inducible promoters on the cell OD₆₀₀ for strains harboring the rare codon-rich markers, the dashed line represents the cell OD₆₀₀ for strain carrying the wild-type kan^R driven by its original promoter from pET-28a. Values and error bars represent the mean and the s.d. (n = 3)

Fig. 5

The amino acids produced by the wild-type and the mutated strains. a l-leucine productions of E. coli mutants selected by the leucine rare codon-rich kan^R. b l-arginine productions of E. coli mutants selected by the arginine rare codon-rich kan^R. c l-serine productions of E. coli mutants selected by the serine rare codon-rich spec^R. d l-arginine productions of C. glutamicum mutants selected by the arginine rare codon-rich kan^R. Values and error bars represent the mean and the s.d. (n = 3). **P < 0.01, ***P < 0.001 as determined by two-tailed t test

Fig. 6

Transcription profiles of BCAA biosynthetic pathways in LP-4 and LP-7. a Transcriptome analysis of LP-4, LP-7, and the wild-type strains. Positive values (red) represent upregulated genes and negative values (blue) represent downregulated genes, which were calculated by the RPKM of LP-4 and LP-7 divided by that of the wild-type strain (in log₂). The top, middle, and bottom panels contain genes in glycolysis, l-leucine biosynthesis and BCAA transportations, respectively. b The BCAA biosynthetic pathways in E. coli, red and blue arrows indicate the up- and downregulated genes, respectively. c qRT-PCR verification of the genes related to l-leucine biosynthesis and transportation. Values and error bars represent the mean and the s.d. (n = 3)