Development strategy of non-GMO organism for increased hemoproteins in Corynebacterium glutamicum: a growth-acceleration-targeted evolution

Abstract

Heme, found in hemoproteins, is a valuable source of iron, an essential mineral. The need for an alternative hemoprotein source has emerged due to the inherent risks of large-scale livestock farming and animal proteins. Corynebacterium glutamicum, regarded for Qualified Presumption of Safety or Generally Recognized as Safe, can biosynthesize hemoproteins. C. glutamicum single-cell protein (SCP) can be a valuable alternative hemoprotein for supplying heme iron without adversely affecting blood fat levels. We constructed the chemostat culture system to increase hemoprotein content in C. glutamicum SCP. Through adaptive evolution, hemoprotein levels could be naturally increased to address oxidative stress resulting from enhanced growth rate. In addition, we used several specific plasmids containing growth-accelerating genes and the hemA promoter to expedite the evolutionary process. Following chemostat culture for 15 days, the plasmid in selected descendants was cured. The evolved strains showed improved specific growth rates from 0.59 h^-1 to 0.62 h^-1, 20% enhanced resistance to oxidative stress, and increased heme concentration from 12.95 µg/g-DCW to 14.22-15.24 µg/g-DCW. Notably, the putative peptidyl-tRNA hydrolase-based evolved strain manifested the most significant increase (30%) of hemoproteins. This is the first report presenting the potential of a growth-acceleration-targeted evolution (GATE) strategy for developing non-GMO industrial strains with increased bio-product productivity.

Keywords: Corynebacterium glutamicum; Adaptive evolution; Hemoproteins; Positive feedback.

Fig. 1

a Growth curves and heme concentrations of C. glutamicum. b Heme concentration of C. glutamicum depends on the specific growth rate (µ). Open circles indicate log (OD₆₀₀), and closed circles indicate heme concentration

Fig. 2

Schematic diagram of GATE. The red neon sign means activation of PhemA. The gray arrow indicates the one direction of normal evolution. The blue arrow is a positive feedback loop formed by the specific plasmid is present

Fig. 3

Schematic diagram of chemostat culture and evolution selection. The chemostat culture was conducted for 15 days. The PhemA-SBP-containing strain was eliminated. After plasmid curing, Evol^CspA, Evol^Pth, and Evol^RamA were selected

Fig. 4

Assessment of oxidative stress resistance and specific growth rate (µ) at the mid-log phase (4–5 h of culture). Resistance to oxidative stress was displayed as intracellular ROS levels. White bars indicate relative ROS levels, and closed circles indicate µ

Fig. 5

Measurement of intracellular heme amount and concentration at the mid-log phase (4–5 h of culture). White bars indicate heme amount, and closed circles indicate heme concentration