Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant.

Min Ju Lee,Kyunghoon Park,Pil Kim,Jihyun F Kim,Jihoon Park

doi:10.3389/fbioe.2020.588070

Abstract

We previously reported a Corynebacterium glutamicum JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide the reverse engineering targets responsible for boosting recombinant protein production and cell reproduction, we compared the genome sequence of the JH41 strain with its ancestral strain. Among the 15 genomic variations, a point mutation was confirmed in the 14 bases upstream of NCgl1959 (encoding a presumed siderophore-binding protein). This mutation allowed derepression of NCgl1959, thereby increasing iron consumption and ATP generation. A point mutation in the structural gene ramA (A239G), a LuxR-type global transcription regulator involved in central metabolism, allowed an increase in glucose consumption. Therefore, mutations to increase the iron and carbon consumption were concluded as being responsible for the enhanced production of recombinant protein and cell reproduction in the evolved host.

Highlights

Many attempts through microbial metabolic engineering have been made to overproduce metabolites by (1) improving the substrate uptake rate, (2) reducing the flux to undesired byproducts, (3) introducing heterologous pathways and optimizing the activity of the enzyme, and (4) balancing product formation in cells, such as secretion of product to the extracellular medium (Yadav et al, 2012)
Because protein is the major macromolecule in cells, the rapidly reproducing JH41 strain was supposed to be able to produce a protein at an enhanced rate
Along with increases in the three ribosomal RNAs (rRNAs), our previous paper reported that most of the messenger RNA (mRNA) encoding ribosomal proteins were upregulated (Supplementary Table 2) (Park et al, 2020). These results enabled us to conclude the internal transcribed spacer (ITS) mutation in rRNA primary transcript supported the fast growth of JH41 by increases in matured rRNAs and ribosomal proteins, and the increase in ribosome components might be a reverse engineering target to enhance the production of recombinant protein even though the genomic introduction of the multiple mutations in the one ITS region among the 6 rRNA operons would be technically difficult

Summary

Introduction

Many attempts through microbial metabolic engineering have been made to overproduce metabolites by (1) improving the substrate uptake rate, (2) reducing the flux to undesired byproducts, (3) introducing heterologous pathways and optimizing the activity of the enzyme, and (4) balancing product formation in cells, such as secretion of product to the extracellular medium (Yadav et al, 2012). Adaptive laboratory evolution (ALE) is a reasonable approach because it copes with the physical defects of strains caused by genetic manipulation and improves the productivity of cell factories (Sandberg et al, 2019; Lee and Kim, 2020). Wang et al reported that the introduction of gntR1 and ramA mutations into Corynebacterium glutamicum increased the growth rate by 37% and the productivity of lysine by 100% (Wang et al, 2018)

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Conclusion