Strain Development by Whole-Cell Directed Evolution

Si, Tong; Lian, Jiazhang; Zhao, Huimin

doi:10.1007/978-3-319-50413-1_7

Cited by 4 publications

(4 citation statements)

References 167 publications

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“…Beyond their interest for natural evolution, questions about how fast and how far evolution proceeds and how these depend on the nature of the evolutionary process, including strength of competition, sizes of population or multiple partially separated sub-populations, etc are also of practical interest as evolution is being used in both bioengineering [37,51] as well as machine learning [48] in order to optimize a wide spectrum of systems.…”

Section: Beyond Weak-mutation Strong-selection Dynamicsmentioning

confidence: 99%

Adaptive walks on high-dimensional fitness landscapes and seascapes with distance-dependent statistics

Agarwala

Fisher

2018

Preprint

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The dynamics of evolution is intimately shaped by epistasis -interactions between genetic elements which cause the fitness-effect of combinations of mutations to be nonadditive. Analyzing evolutionary dynamics that involves large numbers of epistatic mutations is intrinsically difficult. A crucial feature is that the fitness landscape in the vicinity of the current genome depends on the evolutionary history. A key step is thus developing models that enable study of the effects of past evolution on future evolution. In this work, we introduce a broad class of high-dimensional random fitness landscapes for which the correlations between fitnesses of genomes are a general function of genetic distance. Their Gaussian character allows for tractable computational as well as analytic understanding. We study the properties of these landscapes focusing on the simplest evolutionary process: random adaptive (uphill) walks. Conventional measures of "ruggedness" are shown to not much affect such adaptive walks. Instead, the long-distance statistics of epistasis cause all properties to be highly conditional on past evolution, determining the statistics of the local landscape (the distribution of fitness-effects of available mutations and combinations of these), as well as the global geometry of evolutionary trajectories. In order to further explore the effects of conditioning on past evolution, we model the effects of slowly changing environments. At long times, such fitness "seascapes" cause a statistical steady state with highly intermittent evolutionary dynamics: populations undergo bursts of rapid adaptation, interspersed with periods in which adaptive mutations are rare and the population waits for more new directions to be opened up by changes in the environment. Finally, 1

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Section: Beyond Weak-mutation Strong-selection Dynamicsmentioning

confidence: 99%

Adaptive walks on high-dimensional fitness landscapes and seascapes with distance-dependent statistics

Agarwala

Fisher

2018

Preprint

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“…Whole-cell directed evolution, which employs iterative rounds of diversity generation and screening, is considered as a promising strategy to develop desirable production traits at the genome-wide level [ 23 – 25 ]. Genome-wide diversity generation approaches have been developed to be more efficient and well applied, such as irrational methods including UV mutagenesis, EMS chemical mutagenesis, genome shuffling, atmospheric and room temperature plasma (ARTP) mutagenesis, etc., and rational methods including site-directed RNAi, CRISPR-Cas, recombineering, and transposon insertion, etc.…”

Section: Introductionmentioning

confidence: 99%

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

Yuan

Zhou²,

Lin

et al. 2022

Biotechnol Biofuels

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Background Pichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level. Results In this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production. Conclusions We established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

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“…Owing to its simplicity and effectiveness, evolutionary engineering, also known as adaptive laboratory evolution (ALE) or whole‐cell directed evolution, is the preeminent method for both improving key industrial strain traits and analyzing complex tolerance phenotypes that develop under the pressure of artificial selection . In evolutionary engineering, different phenotypes of the same species are due to spontaneous evolutionary variation.…”

Section: Introductionmentioning

confidence: 99%

Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review

Zhou

Zhang

et al. 2019

Biotech and App Biochem

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The diversity of stress responses and survival strategies evolved by microorganism enables them to survive and reproduce in a multitude of harsh environments, whereas the discovery of the underlying resistance genes or mechanisms laid the foundation for the directional enhancement of microbial tolerance to abiotic stresses encountered in industrial applications. Many biological techniques have been developed for improving the stress resistance of industrial microorganisms, which greatly benefited the bacteria on which industrial production is based. This review introduces the main techniques for enhancing the resistance of microorganisms to abiotic stresses, including evolutionary engineering, metabolic engineering, and process engineering, developed in recent years. In addition, we also discuss problems that are still present in this area and offer directions for future research.

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Strain Development by Whole-Cell Directed Evolution

Cited by 4 publications

References 167 publications

Adaptive walks on high-dimensional fitness landscapes and seascapes with distance-dependent statistics

Adaptive walks on high-dimensional fitness landscapes and seascapes with distance-dependent statistics

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review

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