Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress.

Deepanwita Banerjee,Thomas Eng,Vasanth R Singan,Dan Putnam,Aindrila Mukhopadhyay,Corinne D Scown,Jessica Trinh,Ning Sun,Asun Oka,Yusuke Sasaki,Aparajitha Srinivasan,Robin A Herbert,Blake Simmons

doi:10.3389/fbioe.2021.766674

Abstract

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Highlights

Due to process advantages, biological methods for the production of amino acids over chemical synthesis methods fostered the identification of natural glutamine overproducing microbes (Kinoshita et al, 1958)
When C. glutamicum BRCJBEI 1.1.2 is used in conjunction with an IP production pathway, it can produce 300 mg/L IP from pure glucose, but the product titers are near the lower detection limit by GC-FID in the C. glutamicum ATCC 13032 Δmrr strain
For the remainder of this study, we focus on characterizing the genetic differences present in C. glutamicum BRC-JBEI 1.1.2 relative to other closely related C. glutamicum strains that might explain the IP production values between these two strains

Summary

Introduction

Biological methods for the production of amino acids over chemical synthesis methods fostered the identification of natural glutamine overproducing microbes (Kinoshita et al, 1958). Corynebacterium glutamicum has been used successfully to produce specialty glutamine and specialty amino acids to meet global demand. The advent of accessible whole-genome sequencing and mutagenesis methods have enabled a clearer understanding of how specific isolates can overproduce these desired molecules, as well as how they maintain productivity across volumetrically-larger scales (Becker et al, 2018; Pérez-García and Wendisch, 2018; Wolf et al, 2021). Using C. glutamicum to produce non-native metabolites as nextgeneration biofuels is an attractive large-volume market with the potential to reduce global carbon emissions. Genomics of Engineered Corynebacterium glutamicum precursors (reviewed in (Pérez-García and Wendisch, 2018)). Producing IP was improved using optimal pathway homologs, specific media formulation and aeration conditions and an empirically determined carbon/ nitrogen ratio (Sasaki et al, 2019)

Methods

Results

Conclusion