Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

Summary

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

Abstract

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.

Introduction

The current production of amino acids relies heavily on fermentation. However, the titers and yields for most amino acid production strains are below the rising demands of the global amino acid market that is worth billions of dollars^1,2. Obtaining high-performance amino acid overproducers are critical for the upgrade of the amino acid industry.

Traditional strategy to identify amino acid overproducers exploits the competitions between amino acids and their analogs in protein synthesis^3,4. These analogs are able to charge th....

Protocol

1. Construction of the plasmids expressing the rare-codon-rich marker genes

1. Select a marker gene that contains an appropriate number of the common codons for the targeted amino acid.
   NOTE: For L-leucine, the kanamycin resistance gene kan^R, which contains 29 leucine codons, of which 27 are common codons, is used for the construction of the selection system¹³. The gfp gene, which contains 17 common codons out of 19 leucine codons, or the purple protein-encoding gene prancerpurple (ppg), which harbors 14 leucine common codons, is used for the screening system (Supplementary ....

Results

For the selection system, a sharp decrease in OD₆₀₀ for strains harboring the rare-codon-rich antibiotic resistance gene should be observed in comparison to the strain harboring the wild-type antibiotic resistance gene when cultured in a suitable medium (Figure 1a). Under the same conditions, the decrease in cell OD₆₀₀ becomes more obvious as the number of rare codons in the antibiotic resistance gene increases (Figu.......

Discussion

The number of rare codons in the marker genes and the selection or screening medium are critical to inhibit protein expressions from the rare-codon-modified marker genes. If no significant difference can be detected between protein expressions from the wild-type marker genes and their derivatives, increasing the number of rare codons or using a nutrient-limited medium may amplify the differences. However, if the inhibition effect is too strong, the protein expressions may not be recovered even by extra feeding of the cor.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Thermo	51101
EasyPure HiPure Plasmid MiniPrep Kit	Transgen	EM111-01
EasyPure Quick Gel Extraction Kit	Transgen	EG101-01
Gibson assembly master mix	NEB	E2611S
Isopropyl β-D-1-thiogalactopyranoside	Solarbio	I8070
L-leucine	Sigma	L8000
Microplate reader	Biotek	Synergy 2
n-hexane	Thermo	H3061
Phenyl isothiocyanate	Sigma	P1034
PrancerPurple CPB-37-441	ATUM	CPB-37-441
TransStar FastPfu Fly DNA polymerase	Transgen	AP231-01
Triethylamine	Sigma	T0886
Ultra-high performance liquid chromatography	Agilent	1290 Infinity II
Wild type C. glutamicum	ATCC	13032
XL10-Gold E. coli competent cell	Agilent	200314
ZORBAX RRHD Eclipse Plus C18 column	Agilent	959759-902K