Corynebacterium glutamicum Chassis C1*: Building and Testing a Novel Platform Host for Synthetic Biology and Industrial Biotechnology

Abstract

Targeted top-down strategies for genome reduction are considered to have a high potential for providing robust basic strains for synthetic biology and industrial biotechnology. Recently, we created a library of 26 genome-reduced strains of Corynebacterium glutamicum carrying broad deletions in single gene clusters and showing wild-type-like biological fitness. Here, we proceeded with combinatorial deletions of these irrelevant gene clusters in two parallel orders, and the resulting library of 28 strains was characterized under various environmental conditions. The final chassis strain C1* carries a genome reduction of 13.4% (412 deleted genes) and shows wild-type-like growth behavior in defined medium with d-glucose as carbon and energy source. Moreover, C1* proves to be robust against several stresses (including oxygen limitation) and shows long-term growth stability under defined and complex medium conditions. In addition to providing a novel prokaryotic chassis strain, our results comprise a large strain library and a revised genome annotation list, which will be valuable sources for future systemic studies of C. glutamicum.

Less Sacrifice, More Insight: Repeated Low‐Volume Sampling of Microbioreactor Cultivations Enables Accelerated Deep Phenotyping of Microbial Strain Libraries

“…The determined PIs are in good agreement with numerous previous reports (cf. Table S1, Supporting Information), proving that the presented workflow is suitable to replace laborious shake flask or bioreactor experiments for standard growth phenotyping.…”

“…The two genome‐reduced strains carrying the largest gene cluster deletions, PC1_NprE and PC1_53_NprE, showed no significant differences in growth and cutinase production compared to the wild type (Table ). These findings are consistent with previous results, where the background strains (without expression plasmids) showed no differences in defined and enriched CGXII medium . Hence, it can be concluded that there is no observable interaction between the products of deleted genes and cutinase secretion, i.e., all deleted genes could be classified as irrelevant for this purpose.…”

“…The novel workflow was applied for the characterization of a set of recently introduced genome‐reduced strains (GRS) of C. glutamicum that were further engineered toward heterologous cutinase secretion. The GRS carry deletions of all three prophages, two insertion elements and different combinations of larger gene clusters (Table ).…”

“…The biotin‐auxotrophic wild‐type strain C. glutamicum ATCC 13032 as well as further genome‐reduced variants (listed in Table 2) were used in this study …”

Plant synthetic biology: One answer to global challenges

“…While the first artificial genomes were basically copying natural genomes, new approaches aimed at genome reductions to design the optimal minimal cell (“ model chassis ”) for biotechnological production systems. Examples are Mycoplasma mycoides JCVI‐syn3.0 (Hutchison et al ) which has a 50% reduced genome size compared to M. mycoides , the “Minibacillus” with a 36% reduced genome size compared to wildtype Bacillus subtillis (Reuß et al ), or Corynebacterium glutamicum with 13.4% genome reduction (Baumgart et al ). From here, synthetic biology moved on to engineer yeast (Richardson et al ) and mammalian (Martella et al ) artificial chromosomes.…”