Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of Bacillus megaterium.

Thibault Godard,Georg Richter,Ignacio Poblete-Castro,Katharina Riedel,Manfred Rohde,Melanie Wall,Daniela Zühlke,Rainer Krull,Rebekka Biedendieck

doi:10.3389/fbioe.2020.00047

Abstract

For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.

Highlights

Bacillus megaterium is a big rod-shaped Gram-positive soil bacterium that has been used industrially for decades and whose product portfolio is continuously growing
Genes encoding proteins involved in balancing the redox state such as the pyruvate oxidase Pox were found overexpressed (2.5-fold), correlating well with similar observations made for the proteome of cells exposed to at least 1.2 M NaCl (Figure 6)
Since the pox gene showed no difference in expression at 0.6 M NaCl despite the already increased NADH-to-NAD+ ratio, activation of the Pox route seems only indirectly related to the cellular redox state

Summary

Introduction

Bacillus megaterium is a big rod-shaped Gram-positive soil bacterium that has been used industrially for decades and whose product portfolio is continuously growing. It includes enzymes such as α- and β-amylases, penicillin G acylase, xylanase, hydrolases, glycosyltransferases, and cytochrome monooxygenases (Vary et al, 2007; Korneli et al, 2013; Lakowitz et al, 2017; Mayer et al, 2019). The recent sequencing of the complete genome of different B. megaterium strains and the fast development of related omics-techniques have laid the foundations for an in-depth analysis from gene expression to metabolic fluxes, and opened up new possibilities toward its rational genetic engineering (Biedendieck et al, 2011; Eppinger et al, 2011; Liu et al, 2011; Johnson et al, 2015; Freedman et al, 2018)

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Discussion

Conclusion