Table 4 Applications of in vivo continuous evolution in metabolic pathway evolution for chemical production

Coupling ATP and lactate production to growth by engineering lactate production route as the sole anaerobic NADH oxidation route. Selection and enrichment were carried out during fermentation process in flask or fermentation vessels

Natural mutation

Metabolic evolution with LB or NBS medium

Flask or fermentation vessels

A relatively long evolution time is maintained.

The setups are simple

Involvement of rational design in pathway construction can make the process difficult

Production of d-lactate increase from 865 \(\pm\) 36 mmol l⁻¹ to 1071 \(\pm\) 2 mmol l⁻¹

[83]

Metabolic evolution with NBS medium with glucose and betaine (optional)

Production of l-lactate increase from 1228 \(\pm\) 31 mmol l⁻¹ to 1314 \(\pm\) 48 mmol l⁻¹

[85]

Coupling ATP and growth to alanine production and NADH oxidation. Selection and enrichment were carried out during fermentation process in flask or fermentation vessels

Metabolic evolution with NBS or AM1 medium with glucose

Production of l-alanine increase from 181 mmol l⁻¹ to 1279 mmol l⁻¹

[86]

D(-)-lactate hydrogenase fermentation pathway as the sole fermentative pathway coupled with the growth of strain. Selection and enrichment were carried out during fermentation process in flask or fermentation vessels

Metabolic evolution with NBS medium with 9% xylose

Production of ethanol increase from 250 mM to 950 mM

[87]

Coupling growth and glucose fermentation to NADH oxidisation pathway. Selection and enrichment were carried out during fermentation process in flask or fermentation vessels

Metabolic evolution with NBS or AM1 medium with glucose

Production of succinate increase from 108 mM to 699 mM while production of malate increases from 0 mM to 313 mM

[88, 89]

Maltose hydrolase is coupled to couple cell growth to tryptophan production. Selection and enrichment were performed in culture medium with maltose as the sole carbon source

Generation of library with ep-PCR followed by natural mutation 

Artificial metabolite production/cell fitness coupling with maltose

Flask

Continuous evolution without the generation of escapees

Human intervention is required on every cycle of serial dilution

Increase in l-tryptophan titre from 0.5 mg l⁻¹ OD ⁻¹₆₀₀ to 5 mg l⁻¹ OD ⁻¹₆₀₀

[95]

EvolvR is performed to mutate rspE and rspL which is known to modify streptomycin resistance of bacteria. Serial culture was used to realise continuous culture

Genome-targeted mutagenesis with EvolvR

Artificial metabolite production/cell fitness coupling by culturing cells in selective media with xylose as sole carbon source

Flask

High-throughput mutant library generation

Human intervention is required on every cycle of serial dilution

16,000-fold increase in fraction of the population resistant to spectinomycin

[41]

SCRaMbLE was induced for 4 h and the cells were plated on synthetic dropout medium without uracil. Then, 87 coloies were picked at random and grown in selective medium with xylose as sole carbon source. The growth rate was monitored for 5 days

Recombination-based mutagenesis with SCRaMbLE

Artificial metabolite production/cell fitness coupling by culturing cells in selective medium with xylose as sole carbon source

Flask

High-throughput mutant library generation

Human intervention is required on every cycle of serial dilution

Growth rate increase from 0.18 h-1 to 0238 h-1 in culture medium with xylose as the sole carbon source.

[69]

dnaQ PE mutant library is generated and transfected into host cells to generate diversity in host cell. Under selection pressure, only the offspring with adaptive mutation can survive. These mutants can be used in biofuels production

Modified natural mutagenesis with dnaQ proofreading element (PE) mutant library

n-butanol

Flask

Continuous genotype diversification is achieved by transfected PE mutant

Human intervention is required on every cycle of serial dilution.

Limited to specific strain

100-fold increase in survival rate in 2% n-butanol compared to wild type after 18 transfers.

[125]

Acetate

eightfold increase in survival rate in 0.1% acetate compared to wild type after 12 transfers.

PACE was performed to evolved the product protein aspartate kinase III, while l-lysine was used as a selection pressure for selection due to its inhibitory properties to aspartate kinase III

Modified natural mutagenesis with dnaQ926

l-lysine

Chemostat

Continuous evolution without any human intervention

Limited to specific strain which can be infected by bacteriophage

Absolute activity with 50 mM of lysine increase from 0.1 to 0.3; Less than 20% drop in relative activity when inhibited by 100 mM lysine

[130]