Evolutionary engineering of industrial microorganisms-strategies and applications

Zhu, Zhengming; Zhang, Juan; Ji, Xiaomei; Fang, Zhen; Wu, Zhimeng; Chen, Jian; Du, Guocheng

doi:10.1007/s00253-018-8937-1

Cited by 55 publications

(28 citation statements)

References 126 publications

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“…In order to select for superior strain variants, industrial production strains are often subjected to directed evolution techniques such as random mutagenesis, protoplast fusion or genome shuffling 20 . Although these techniques are less dependent on biological information and available genetic tools, they suffer from being non-directional, having the risk of improving one trait to the detriment of others 20 , 21 .…”

Section: Introductionmentioning

confidence: 99%

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Cámara

Lenitz

Nygård

2020

Sci Rep

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A cRiSpR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12 elena cámara, ibai Lenitz & Yvonne nygård * Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains. The yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for industrial applications ranging from wine and beer fermentations to the production of biofuels and high-value metabolites 1,2. However, some of the current production processes are compromised by low yields and productivities, thus further optimization is required 3. In particular, the production of second-generation bioethanol and other biochemicals from lignocellulosic biomass, which provides an alternative to oil-based chemicals, suffers from sub-optimal productivity 4. During the hydrolysis of the raw material, inhibitory compounds (e.g. organic acids and aromatic aldehydes) are formed or released, compromising the microbial performance 5. While quite some work has been done on elucidating genes required for tolerance, much less work has been done on improving tolerance towards stress by altering expression of genes 6. Some previous studies demonstrated deletion 7-9 or overexpression 10-14 of endogenous genes to improve tolerance of S. cerevisiae towards inhibitors commonly found in lignocellulosic hydrolysates. However, most of the published work focuses on improving the tolerance of laboratory yeast strains that generally have weaker tolerance to stress 6 , while translation of beneficial modifications to more robust, industrial strains often is very challenging. The choice of yeast strain to be engineered is crucial for the successful implementation of the engineered phenotype in an industrial production process 15. Yeast strains used in industrial processes tend to be genetically diverse, since they usually arise from hybridization between different species 16. Hence...

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Section: Introductionmentioning

confidence: 99%

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Cámara

Lenitz

Nygård

2020

Sci Rep

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“…The plasma jet generated by ARTP is controlled by the discharged power and gas flow, and its composition mainly includes excited helium atoms, excited oxygen atoms, excited hydrogen atoms, and free •OH radicals (Zhang et al, 2014;Winter et al, 2015;Zhang et al, 2015;Yu et al, 2019). Due to its low voltage radio-frequency power source, low temperature plasma, equal uniform plasma jet, easy operation, and high safety, ARTP has become increasingly popular among microbial breeding operators for its extensive applications (Wang et al, 2010;Zhang et al, 2014;Yang et al, 2019;Zhou et al, 2019). Li et al (2008) have identified the mechanism of radio-frequency and atmospheric-pressure glow discharges (APGD) directly acted on plasmid DNA and oligonucleotides.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, APGD was successfully used for the breeding of Streptomyces avermitilis to enhance the production of avermectins ( Wang et al, 2010 ). To date, ARTP, which was developed based on the concept of APGD, has become an efficient breeding method in the field of industrial microbiology ( Zhang et al, 2014 , 2015 ; Wu et al, 2015 ; Zhu et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Enhancement of Acid Protease Activity of Aspergillus oryzae Using Atmospheric and Room Temperature Plasma

Shu

Yang

et al. 2020

Front. Microbiol.

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Atmospheric and room temperature plasma (ARTP) system is a novel and efficient mutagenesis protocol for microbial breeding. In this study, ARTP was employed to treat spores of Aspergillus oryzae strain 3.042 for selection of high acid protease producers. With an irradiation time of 150 s at the lethal rate of 90%, 19 mutants with higher acid protease activity were initially selected based on different mutant colony morphology and ratio of the clarification halo of protease activity to the colony diameter. Measurements of the acid protease activity revealed that mutant strain B-2 is characterized by a steady hereditary stability with increased acid protease, neutral protease and total protease activities of 54.7, 17.3, and 8.5%, respectively, and decreased alkaline protease activity of 8.1%. In summary, the identified mutant strain B-2 exhibits great potential for the enhancement of the insufficient acid protease activity during the middle and later stages of soy sauce fermentation.

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“…Several strategies have been proposed to increase the acid-stress tolerance of bacterial strains. Evolutionary engineering strategies are extensively used to improve the acid tolerance of microbial cells [8]. The acid tolerance of Lactobacillus casei Zhang has been shown to be increased by adaptive evolution, and the evolved mutant exhibited a 318-fold higher survival rate than that of the parent strain at pH 3.3 for 3 h [9].…”

Section: Introductionmentioning

confidence: 99%

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

Zhu

Yang

et al. 2019

Microb Cell Fact

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Background Microbial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis . Results Overexpressing the rbsA , rbsB , msmK , and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins ( csp ), fatty acid biosynthesis ( fabH ), and coenzyme A biosynthesis ( coaD ) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway ( pyrCBDEK ) and glycine or betaine transport process ( busAA and busAB ) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress. Conclusions This study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance. Electronic supplementary material The online version of this article (10.1186/s12934-019-1188-8) contains supplementary material, which is available to authorized users.

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Evolutionary engineering of industrial microorganisms-strategies and applications

Cited by 55 publications

References 126 publications

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Enhancement of Acid Protease Activity of Aspergillus oryzae Using Atmospheric and Room Temperature Plasma

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

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