Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Abstract Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.

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Current status on metabolic engineering for the production of l-aspartate family amino acids and derivatives

Bioresource Technology ◽

10.1016/j.biortech.2017.05.145 ◽

2017 ◽

Vol 245 ◽

pp. 1588-1602 ◽

Cited By ~ 33

Author(s):

Yanjun Li ◽

Hongbo Wei ◽

Ting Wang ◽

Qingyang Xu ◽

Chenglin Zhang ◽

...

Keyword(s):

Amino Acids ◽

Metabolic Engineering ◽

Current Status ◽

Amino Acids And Derivatives ◽

Aspartate Family

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Multiplex Design of the Metabolic Network for Production of L-Homoserine in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01477-20 ◽

2020 ◽

Vol 86 (20) ◽

Cited By ~ 1

Author(s):

Peng Liu ◽

Bo Zhang ◽

Zhen-Hao Yao ◽

Zhi-Qiang Liu ◽

Yu-Guo Zheng

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Shake Flask ◽

Isocitrate Lyase ◽

Metabolic Effects ◽

Homoserine Dehydrogenase ◽

Metabolomics Data ◽

Systematic Analysis ◽

Cell Factories ◽

Shake Flask Cultivation

ABSTRACT l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose. IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.

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Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

10.1101/2020.04.14.041889 ◽

2020 ◽

Author(s):

Hong Liang ◽

Xiaoqiang Ma ◽

Wenbo Ning ◽

Yurou Liu ◽

Anthony J. Sinskey ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Carbon Source ◽

Sole Carbon Source ◽

Structural Similarity ◽

List Type ◽

Acetyl Coa ◽

E Coli ◽

Utilization Pathway ◽

Aspartate Family

AbstractEngineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.HighlightsEngineered Escherichia coli strains to grow on ethanol as sole carbon sourceDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA)Converted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol)Discovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol

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A Family of Stability Determinants in Pathogenic Bacteria

Journal of Bacteriology ◽

10.1128/jb.180.23.6415-6418.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6415-6418 ◽

Cited By ~ 40

Author(s):

Finbarr Hayes

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Haemophilus Influenzae ◽

Vibrio Cholerae ◽

Pathogenic Bacteria ◽

Plasmid Stability ◽

Overlapping Genes ◽

Morganella Morganii ◽

E Coli ◽

Segregational Stability

ABSTRACT A novel segregational stability system was identified on plasmid R485, which originates from Morganella morganii. The system is composed of two overlapping genes, stbD andstbE, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the stbDE genes were identified on the enterotoxigenic plasmid P307 from Escherichia coli and on the chromosomes of Vibrio cholerae andHaemophilus influenzae biogroup aegyptius. The former two homologs also promote plasmid stability in E. coli. Furthermore, the stbDE genes share homology with components of the relBEF operon and with thednaT gene of E. coli. The organization of thestbDE cassette is reminiscent of toxin-antitoxin stability cassettes.

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