Using biopolymer bodies for encapsulation of hydrophobic products in bacterium

Liu, Yurou; Low, Zhen Jie; Ma, Xiaoqiang; Hong, Liang; Sinskey, Anthony J.; Stephanopoulos, Gregory; Zhou, Kang

doi:10.1016/j.ymben.2020.04.006

Cited by 16 publications

(15 citation statements)

References 37 publications

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“…To apply CRASH protocol, we have tested 11 gene knockouts and iteratively knockout 7 genes from BL21 genome to improve lycopene production. Recently, several works have demonstrated that increasing the storage capacity of the production microorganisms is beneficial to lycopene production [24,25,34]. Here, we demonstrated two more strategies to increase the storage capacity: first is to increase cell size and second is to enhance the neutral lipid production.…”

Section: Discussionmentioning

confidence: 62%

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Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Shukal

Lim

Zhang

et al. 2021

Preprint

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BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.

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Section: Discussionmentioning

confidence: 62%

“…5a). Since lycopene is an intracellular compound, increasing cellular storage capacity may provide a favourable pull to enhance the specific yield of lycopene [24,25]. Thus, we hypothesized that by altering the cell size might increase the storage capacity.…”

Section: Increasing E Coli Sizementioning

confidence: 99%

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Shukal

Lim

Zhang

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…5 a). Since lycopene is an intracellular compound, increasing cellular storage capacity may enhance the specific yield of lycopene [ 24 , 25 ]. Thus, we hypothesized that altering the cell size might increase the storage capacity.…”

Section: Resultsmentioning

confidence: 99%

“…To apply the CRASH protocol, we tested 11 gene knockouts and iteratively knocked out 7 genes from the BL21 genome to improve lycopene production. Several recent studies have demonstrated that increasing the storage capacity of the microorganisms is beneficial to lycopene production [ 24 , 25 , 34 ]. Here, we demonstrated two more strategies to increase the storage capacity: firstly to increase the cell size and secondly to enhance the neutral lipid production.…”

Section: Discussionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

et al. 2022

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Background The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked. Results In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold. Conclusions Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.

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“…To overcome the limited storage capacity in the tobacco chloroplast as a native host system, the droplet-forming hydrophobic protein derived from oleosin was co-produced with hydrophobic squalene in the tobacco chloroplast, used as a synthetic co-compartment, to increase the intracellular storage of squalene . Recently, poly(3-hydroxybutyrate) (PHB) was co-produced for in vivo heterologous storage to improve lycopene production (120 mg/L) from isopentenol …”

Section: Introductionmentioning

confidence: 99%

Microbial Bioprocess for Extracellular Squalene Production and Formulation of Nanoemulsions

Park

Kang

Woo

2021

ACS Sustainable Chem. Eng.

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Sustainable production of natural resources requires a clean manufacturing process with clean raw materials due to the risk of environmental contaminations. Microbial cell factories have enabled the production of natural products including hydrophobic chemicals. However, the microbial cells limit the production titer because of the limited intracellular storage capacity and no transport across the cell membrane. Herein, we engineered Corynebacterium glutamicum to produce hydrophobic squalene as a model product. Notably, squalene was secreted in the defined culture medium without in situ extractions. As a result, the secreted squalene was related to the glycolipid association with synthesized squalene in the cell envelope and a facilitated secretion occurred during cell division. Protein engineering and pathway engineering of C. glutamicum enhanced the total squalene (tSQ) production, where fed-batch fermentation resulted in 1406 ± 16 mg/L tSQ production (32% tSQ in the medium). Promoting extraction with dodecane overlay, bio-squalene nanoemulsions were formulated and the creaming effects were analyzed for cosmetic application. Combining metabolic pathway engineering and facilitated secretion in microbial cells will be a good strategy to produce sustainable hydrophobic chemicals.

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Using biopolymer bodies for encapsulation of hydrophobic products in bacterium

Cited by 16 publications

References 37 publications

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

Microbial Bioprocess for Extracellular Squalene Production and Formulation of Nanoemulsions

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