High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration

Huang, Shuangcheng; Geng, Anli

doi:10.1016/j.jbiotec.2020.01.014

Cited by 20 publications

(11 citation statements)

References 32 publications

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“…Another method suited to highcopy number integration is CRISPR mediated δ-integration. This method was used in two recent studies to integrate multiple genes in to δ sites, yielding 18 (Shi et al, 2016) and 25 copies (Huang and Geng, 2020), respectively.…”

Section: Discussionmentioning

confidence: 99%

“…However, a maximum copy number of four was achieved for both δ and rDNA site integration. The coupling of CRISPR/Cas9 with rDNA, CMGE (Wang et al, 2018), δ-integration (Shi et al, 2016;Huang and Geng, 2020) or CRITGI (Hanasaki and Masumoto, 2019) resulted in substantial improvements in integration efficiency and copy number. The use of a combination of CRISPR-mediated approaches therefore has the potential to improve isobutanol titers further.…”

Section: Rdna Clusters and Its Coupling With Crispr/cas9mentioning

confidence: 99%

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Multiplex Genome Engineering Methods for Yeast Cell Factory Development

Malcı

Walls

Rios‐Solis

2020

Front. Bioeng. Biotechnol.

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As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker's yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of preplaced gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.

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Section: Discussionmentioning

confidence: 99%

Section: Rdna Clusters and Its Coupling With Crispr/cas9mentioning

confidence: 99%

Multiplex Genome Engineering Methods for Yeast Cell Factory Development

Malcı

Walls

Rios‐Solis

2020

Front. Bioeng. Biotechnol.

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“…Metabolic engineering can also control the formation of NADH‐dependent by‐products, prioritizing the major pathway for 2,3‐BDO formation over the parallel metabolic pathways 30 . The main modified and recently isolated microorganisms reported in the evaluated documents were: Klebsiella pneumoniae , 31,32 K. oxytoca , 33–35 Paenibacillus polymyxa , 23,36,37 Bacillus subtillis , 38 Clostridium sp., 39,40 Enterobacter cloacae , 41,42 Escherichia coli , 43,44 Raoultella sp., 45,46 Zymobacter sp., 47 Pichia pastoris sp., 24 B. licheniformis , 48 B. amyloliquefaciens , 49,50 Serratia marcescens , 51,52 and Saccharomyces cerevisiae 3,53 . Cellular modifications also aim at process safety, which is considered to be another important requirement for large‐scale 2,3‐BDO production.…”

Section: Resultsmentioning

confidence: 99%

Technological development of the bio‐based 2,3‐butanediol process

Tinôco

Borschiver

Coutinho

et al. 2020

Biofuels Bioprod Bioref

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The biotechnological production of 2,3‐butanediol (2,3‐BDO) has economic and environmental advantages over the chemical route, although its large‐scale implementation has not yet been consolidated. To establish the technological maturity of the bio‐based 2,3‐BDO process, and the factors limiting its industrial use, a technology roadmap was built from patents and scientific articles, in which four drivers were identified: production process, recovery process, product, and application. The production process driver was reported in 53.25% of documents, notably including studies on substrates, engineered microorganisms, efficient production methods, and nutritional supplementation. The recovery process driver was investigated in 23.5% of studies, with conventional distillation and solvent extraction being the main technologies performed on a large scale. Product and application drivers accounted for 9.25% and 14% of studies, respectively, highlighting the chemical intermediates, plastics, food additives, and cosmetics market segments. A limited number of companies like LanzaTech and GS Caltex Corporation already produce industrially bio‐based 2,3‐BDO, while academic centers such as the Yancheng Institute of Technology are focused to the laboratory scale with technologies to be implemented in the long term. This study can therefore help to establish and gain an understanding of the necessary strategies to consolidate the industrial production of bio‐based 2,3‐BDO in the future. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

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“…It should be noted that longer HAs (200 bp and 400 bp) did not increase the integration efficiency (Figure 2c and Figure S6), and some reports also observed a similar phenomenon in HR meditated genomic editing. 26,27 It is speculated that the integration efficiency is influenced in combination by multiple factors, such as homologous recombination (HR) ability 28 or donor DNA transformation efficiency, 29 and these factors could lead to a variation in the integration efficiency. Flexibility and efficiency are two key factors considered in multiplex integration.…”

Section: Recruitment Of Rad51 For Enhanced Integrationmentioning

confidence: 99%

CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae

et al. 2023

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Genomic integration is the preferred method for gene expression in microbial industrial production. However, traditional homologous recombination based multiplexed integration methods often suffer from low integration efficiency and complex experimental procedures. Here, we report a CRISPR/Cas9 based multiplexed integration (CMI) system in Saccharomyces cerevisiae, which can achieve quadruple integration at an individual locus without pre-engineering the host. A fused protein, Cas9-Brex27, was used as a bait to attract Rad51 recombinase to the proximity of the double-strand breaks introduced by the CRISPR/Cas9 system. The efficiency of quadruple integration was increased to 53.9% with 40 bp homology arms (HAs) and 78% with 100 bp HAs. CMI was applied to integrate a heterologous mogrol biosynthetic pathway consisting of four genes in a one-step transformation and offered an efficient solution for multiplexed integration. This method expands the synthetic biology toolbox of S. cerevisiae.

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High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration

Cited by 20 publications

References 32 publications

Multiplex Genome Engineering Methods for Yeast Cell Factory Development

Multiplex Genome Engineering Methods for Yeast Cell Factory Development

Technological development of the bio‐based 2,3‐butanediol process

CMI: CRISPR/Cas9 Based Efficient Multiplexed Integration in Saccharomyces cerevisiae

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