Isolation and characterization of a stable l-arginine producer from continuous culture broth of Corynebacterium acetoacidophilum

Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production.

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“…1 ab—case 3), e.g. as seen in early chemostat-based studies [ 2 , 55 ]. In addition to changes driven by cell heterogeneities (e.g.…”

Section: Non-genetic and Genetic Heterogeneities Affect Bioprocess Pementioning

confidence: 98%

The future of self-selecting and stable fermentations

Rugbjerg

Olsson

2020

Journal of Industrial Microbiology and Biotechnology

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“…C. glutamicum arginine producers used for comparative sequence analysis were strains A-27, I-30, and D-77, which were derived through multiple rounds of mutagenesis from wild-type ATCC 13870, ATCC 13032, and KY10025, respectively (2,17). Strain ATCC 13870 was previously classified as Corynebacterium acetoacidophilum, but based on recent molecular taxonomic studies it is currently reclassified in the original species, C. glutamicum (13).…”

Section: Methodsmentioning

confidence: 99%

“…After growth to early stationary phase at 30°C on a rotary shaker, the seed broth was transferred into a 5-liter jar fermentor containing 1,000 ml of RPG1 medium. After the sugar initially added was consumed, a solution containing (per liter) 500 g of glucose, 30 g of (NH 4 2 O, 20 mg of ␤-alanine, 20 mg of nicotinic acid, 20 mg of thiamine-HCl, and 0.2 mg of D-biotin was continuously fed until the total amount of glucose in the medium reached 572 g. The feeding rate of the solution was controlled to maintain the glucose concentration in the medium at a low concentration (below 0.5%). The culture was basically performed with an agitation speed of 800 rpm, with aeration at 2 liter/min, and at 30°C or 38°C.…”

Section: Methodsmentioning

confidence: 99%

“…When required, kanamycin was added at the final concentration of 20 g/ml for BY plates. RG2 medium used for production in a 300-ml flask consisted of (per liter) 60 g of glucose, 5 g of corn steep liquor, 30 g of (NH 4 ) 2 Cultivations for arginine/citrulline production. For flask fermentations, a 2.0-ml sample of the seed culture grown to early stationary phase at 30°C in BYG medium (containing 1.0% glucose in medium BY) was inoculated into 20 ml of RG2 medium in a 300-ml flask and cultivated aerobically at 30°C or 38°C until the sugar was consumed.…”

Section: Methodsmentioning

confidence: 99%

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Reengineering of a Corynebacterium glutamicum l -Arginine and l -Citrulline Producer

Ikeda

Mitsuhashi

Tanaka

et al. 2009

Appl Environ Microbiol

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Toward the creation of a robust and efficient producer of L-arginine and L-citrulline (arginine/citrulline), we have performed reengineering of a Corynebacterium glutamicum strain by using genetic information of three classical producers. Sequence analysis of their arg operons identified three point mutations (argR123, argG92 up , and argG45) in one producer and one point mutation (argB26 or argB31) in each of the other two producers. Reconstitution of the former three mutations or of each argB mutation on a wild-type genome led to no production. Combined introduction of argB26 or argB31 with argR123 into a wild type gave rise to arginine/citrulline production. When argR123 was replaced by an argR-deleted mutation (⌬argR), the production was further increased. The best mutation set, ⌬argR and argB26, was used to screen for the highest productivity in the backgrounds of different wild-type strains of C. glutamicum. This yielded a robust producer, RB, but the production was still one-third of that of the best classical producer. Transcriptome analysis revealed that the arg operon of the classical producer was much more highly upregulated than that of strain RB. Introduction of leuC456, a mutation derived from a classical L-lysine producer and provoking global induction of the amino acid biosynthesis genes, including the arg operon, into strain RB led to increased production but incurred retarded fermentation. On the other hand, replacement of the chromosomal argB by heterologous Escherichia coli argB, natively insensitive to arginine, caused a threefoldincreased production without retardation, revealing that the limitation in strain RB was the activity of the argB product. To overcome this, in addition to argB26, the argB31 mutation was introduced into strain RB, which caused higher deregulation of the enzyme and resulted in dramatically increased production, like the strain with E. coli argB. This reconstructed strain displayed an enhanced performance, thus allowing significantly higher productivity of arginine/citrulline even at the suboptimal 38°C.We have shown reverse engineering of a high-production strain of Corynebacterium glutamicum by using L-lysine fermentation as a model (10,11,21). The characteristic that the methodology aims at is the robustness of the resulting strain. The classical approach, based on random mutation and selection, sacrifices the native robustness of an organism in exchange for enhancing the production abilities to the limits. The high production abilities and delicate constitutions of classical industrial producers are the merits and demerits of the classical approach. Such an inevitable consequence of the classical approach could be understood also from the fact that more than 1,000 mutations have accumulated in the genome of an industrial L-lysine producer of C. glutamicum (11). We examined those mutations and identified mutations relevant to Llysine production. Subsequent assembly of the useful mutations in a robust wild-type strain was shown to substantially improve producer per...

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“…Production loads typically arise from metabolic depletions ( 6 ) in addition to toxicities from pathway intermediates and end products ( 7 ). Such production loads can cause declining yields due to genetic instabilities and eventually lead to the complete loss of production, posing substantial challenges to the scale-up of bio-based processes ( 8 – 10 ) and applicability of continuous fermentations ( 11 , 12 ). Optimization of long-term genetic strain stability is therefore a necessary and research-intensive process toward commercialization of bioprocesses.…”

mentioning

confidence: 99%

Synthetic addiction extends the productive life time of engineered Escherichia coli populations

Rugbjerg

Sarup-Lytzen

Nagy

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

114

108

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SignificanceBioproduction of chemicals offers a sustainable alternative to petrochemical synthesis routes by using genetically engineered microorganisms to convert waste and simple substrates into higher-value products. However, efficient high-yield production commonly introduces a metabolic burden that selects for subpopulations of nonproducing cells in large fermentations. To postpone such detrimental evolution, we have synthetically addicted production cells to production by carefully linking signals of product presence to expression of nonconditionally essential genes. We addict Escherichia coli cells to their engineered biosynthesis of mevalonic acid by fine-tuned control of essential genes using a product-responsive transcription factor. Over the course of a long-term fermentation equivalent to industrial 200-m3 bioreactors such addicted cells remained productive, unlike the control, in which evolution fully terminated production.

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Isolation and characterization of a stable l-arginine producer from continuous culture broth of Corynebacterium acetoacidophilum

Cited by 16 publications

References 11 publications

The future of self-selecting and stable fermentations

The future of self-selecting and stable fermentations

Reengineering of a Corynebacterium glutamicum l -Arginine and l -Citrulline Producer

Synthetic addiction extends the productive life time of engineered Escherichia coli populations

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