Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon

Har, Jie Ren Gerald; Agee, Alec; Bennett, R. Kyle; Papoutsakis, Eleftherios T.; Antoniewicz, Maciek R.

doi:10.1007/s00253-020-11058-0

Cited by 14 publications

(9 citation statements)

References 28 publications

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“…The same group further investigated the effects of activating amino acid biosynthetic pathways on methanol assimilation and methylotrophic growth of E. coli . Based on the 13 C‐methanol labelling data, several amino acids including l ‐histidine, l ‐isoleucine, l ‐leucine, l ‐lysine, l ‐methionine, l ‐phenylalanine, l ‐threonine, l ‐tyrosine and l ‐valine were identified as the limiting amino acids for E. coli growth on methanol (Bennett et al ., 2020a; Bennett et al ., 2021; Har et al ., 2021). Although much progress has been made in studying the biosynthesis of limiting amino acids from methanol–carbon in methylotrophic E. coli , few studies focus on that in methylotrophic C. glutamicum .…”

Section: Resultsmentioning

confidence: 99%

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

Fan

Wang

Qian

et al. 2021

Microbial Biotechnology

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Methanol is a promising feedstock for biomanufacturing of fuels and chemicals. Although efforts have been made to engineer platform microorganisms for methanol bioconversion, the substrate uptake and cell growth rates on methanol are still unsatisfactory, suggesting certain limiting factors remain unsolved. Herein, we analysed the global metabolic regulation changes between an evolved methanol-dependent Corynebacterium glutamicum mutant and its ancestral strain by transcriptome analysis. Many genes involved in central metabolism including glycolysis, amino acid biosynthesis and energy generation were regulated, implying the adaptive laboratory evolution reprogrammed the cellular metabolism for methanol utilization. We then demonstrated that nitrate could serve as a complementary electron acceptor for aerobic methanol metabolism, and the biosynthesis of several amino acids limited methylotrophic growth. Finally, the sedoheptulose bisphosphatase pathway for generating methanol assimilation acceptor was found effective in C. glutamicum. This study identifies limiting factors of methanol metabolism and provides engineering targets for developing superior synthetic methylotrophs.

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Section: Resultsmentioning

confidence: 99%

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

Fan

Wang

Qian

et al. 2021

Microbial Biotechnology

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“…However, the evolved strain was enabled to grow on threonine without methanol. After cultivation in a methanol‐containing medium coupled with the reduction of threonine concentration for several passages, the growth rate of strain was increased twofold 69 . After the application of insert sequence (IS)‐mediated copy number variations (CNVs) and balance of metabolic flux, the reprogrammed E. coli followed by laboratory evolution solved the hurdle of DNA‐protein crosslinking (DPC), which showed efficient methanol utilization, giving a final OD 600 of 1.9 and consumption of 120 m mol L −1 MeOH 70 …”

Section: Adaptive Laboratory Evolutionmentioning

confidence: 99%

Strategies to improve the stress resistance of Escherichia coli in industrial biotechnology

Tao

Wang

et al. 2022

Biofuels Bioprod Bioref

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The use of industrial Escherichia coli strains for the production of bio‐based chemicals offers a promising and sustainable solution to a growing scarcity of non‐renewable resources and contributes to the protection of the environment. However, many limitations, such as inhibition from toxic substrates, hyperosmosis, toxic by‐products, and other adverse fermentation conditions, prevent strains from attaining a stable cell growth rate, efficient metabolic flux, and satisfactory productivity. There has thus been much effort to obtain E. coli strains that exhibit robust growth and productivity with enhanced tolerance of extreme operating conditions. In this review, we gained insights into recent studies in improving tolerance of E. coli and summarized four kinds of engineering methods: modifying regulation factors, regulating membrane structure, optimizing biosynthetic pathways, and applying adaptive evolution. The combination of different engineering strategies therefore provided a broader platform for robustness of industrial E. coli, making further steps to achieving more advantageous biosynthesis. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd

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“…Adaptive laboratory evolution (ALE) is a widespread used strain-improvement method in which bacteria are continually cultured in stressful conditions for several generations to improve fitness by accumulating beneficial mutations. , ALE was successfully applied to improve bacterial growth, , substrate consumption, − strain robustness, − as well as the titer, yield, and productivity (TRY) of products. , It is also often combined with metabolic engineering and synthetic biology. , …”

Section: Introductionmentioning

confidence: 99%

Improving Furfural Tolerance of Escherichia coli by Integrating Adaptive Laboratory Evolution with CRISPR-Enabled Trackable Genome Engineering (CREATE)

Zheng

Kong

Luo

et al. 2022

ACS Sustainable Chem. Eng.

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Genetic diversity is an important factor affecting the efficiency of adaptive laboratory evolution (ALE). The recent development of precise tools and strategies for genomic engineering has greatly accelerated mutant library construction for ALE. Here, a global regulator library based on the CRISPR-enabled trackable genome engineering (CREATE) technology was first used to accelerate adaptive evolution for improved furfural tolerance in Escherichia coli, and the furfural tolerance was increased approximately 2-fold in the genetically diversified CREATE-based strains. The evolved strain tolerated up to 4.7 g/L furfural and also showed marked cross-tolerance to NaCl, isobutanol, butanol, ethanol, and high temperature. Whole-genome sequencing and mutant reconstruction analysis revealed for the first time that rpoB P153L mutation leads to greatly increased furfural tolerance. The expression of genes coding central carbon and energy metabolism was significantly altered according to transcriptome analysis. In particular, it was confirmed for the first time that the knockout of sRNA sgrS and the overexpression of sRNA arrS significantly increased furfural tolerance. This study provides evidence that combined ALE and the CREATE technology can not only obtain highly efficient strains with favorable mutation combinations but also accelerate ALE by providing much greater genomic and functional diversity.

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Adaptive laboratory evolution of methylotrophic Escherichia coli enables synthesis of all amino acids from methanol-derived carbon

Cited by 14 publications

References 28 publications

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

Transcriptome analysis reveals the roles of nitrogen metabolism and sedoheptulose bisphosphatase pathway in methanol‐dependent growth of Corynebacterium glutamicum

Strategies to improve the stress resistance of Escherichia coli in industrial biotechnology

Improving Furfural Tolerance of Escherichia coli by Integrating Adaptive Laboratory Evolution with CRISPR-Enabled Trackable Genome Engineering (CREATE)

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