Application of CRISPR technology to the high production of biopolymers

CHAPTER

Application of CRISPR technology to the high production of biopolymers

7

Hyo Jin Kim1, 2, Timothy Lee Turner3 1

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea; 2Institutes of Green Bio Science and Technology, Seoul National University, Pyeongchang, Gwangwon-do, Republic of Korea; 3Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States

1. Introduction CRISPR/Cas9 technology has had a great impact on biology, medicine, microbiology, and food microbiology. In particular, the highly sophisticated base pairelevel gene editing technique enabled more precise manipulation of genes to allow for the insertion or removal of several genes quicker and easier than conventional DNA manipulation techniques. Additionally, the ease of deletion of gene targets in polyploidy organisms made a major improvement on research in the life sciences across a variety of eukaryotic systems. Ultimately, CRISPR/Cas9 will promote the development of the biological sciences for a variety of organisms and will facilitate the production of numerous industrially relevant substances, particularly biopolymers. This chapter covers microbial metabolic engineering via the CRISPR/Cas9 technology and focuses on the application of the CRIPSR/Cas9 technology to create various types of biopolymers, including exopolysaccharides (EPSs), produced from food microorganisms.

2. Application of CRISPR/Cas9 in various research The purpose of the CRISPR (clustered regularly interspaced short palindromic repeats) sequence, found in the genomes of bacteria and archaea, remained unknown when it was first discovered by Yoshizumi Ishino at Osaka University, but the function was uncovered years later by scientists at Danisco Company (Barrangou et al., 2007; Ishino et al., 1987). Numerous domesticated bacteria widely used in fermentation and biotechnology processes are often susceptible to phage attack. Barrangou et al. (2007) found that CRISPR together with associated cas genes were involved in resistance against phages, and they showed that the phage resistance indeed was conferred on the host by the CRISPR sequences (Barrangou et al., 2007). In 2012, Emmanuelle Charpentier at Umea˚ University, who is now affiliated with the Max Planck Institute for Infection Biology in Berlin, in collaboration with Jennifer Doudna at the University of California (UC) in Berkeley, has shown that Biopolymer-Based Formulations. https://doi.org/10.1016/B978-0-12-816897-4.00007-2 Copyright © 2020 Elsevier Inc. All rights reserved.

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(A)

Double-strand breakage

Gene disruption

(B)

Double-strand breakage

Gene addition

FIGURE 7.1 (A) Programming Cas9 with crRNA (CRISPR RNA):tracrRNA (transactivating crRNA). (B) Programming Cas9 with single-guide RNA.

the action of dual-RNA (crRNAs and transactivating crRNAs, Fig. 7.1A) and endonuclease Cas9 can generate a double-stranded breakage in target DNA (Jinek et al., 2012). They emphasized the fact that this tool could edit genomes precisely with great ease, and this proved to be true later in other studies (Ceasar et al., 2016). This programmable, dual-RNA-guided DNA endonuclease was found to be much more convenient and widely applicable than conventional restriction enzyme systems, TALEN (transcription activatorelike effector nuclease) nuclease, or zinc-finger nuclease. In addition, the George Church group at Harvard University and his former postdoc Feng Zhang’s group of the Broad Institute succeeded in simultaneously applying the CRISPR/Cas9 system to human cells (Cong et al., 2013; Mali et al., 2013). In particular, the Church group generated the fusion of dual-RNA to enable a more convenient system (Fig. 7.1B) (Mali et al., 2013). The success of this mammalian system facilitated manipulation of eukaryotic genetics, which was previously difficult and labor-intensive, and allowed subsequent CRISPR studies of fungi, fishes, insects, plants, and animals in various fields (Gantz and Akbari, 2018; DiCarlo et al., 2013; Vyas et al., 2015; Hwang et al., 2013; Jiang et al., 2013; Guo and Li, 2015). More importantly, the application of the CRISPR/Cas9 technique for gene therapy has had tremendous medical implications for treating patients with genetic disorders. People suffering from genetic disorders due to mutations of a critical gene for normal cellular functions could potentially be rescued by use of the CRISPR/Cas9 technique by rendering a correction of the mutated

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gene sequence. A similar approach has recently shown the possibility of AIDS treatment by introducing a mutation in a coreceptor for HIV entry to confer resistance against HIV infection (Schumann et al., 2015). This is an important improvement in AIDS research, as this outcome would be truly difficult without the precision afforded by the CRISPR/Cas9 technology. However, further development of CRISPR/Cas9 technology will be needed before it is ready for widespread use in human disease treatment. Using the CRISPR/Cas9 technique enables knock-in and knock-out of target genes. The target gene can be disrupted, or a heterologous gene (or an endogenous overexpression target) can be introduced through the CRISPR/Cas9 technique. The knock-out of the target gene by CRISPR/Cas9 is driven by a molecular mechanism called nonhomologous end joining (NHEJ, Fig. 7.2A). On the other hand, the gene knock-in of the CRISPR/Cas9 technique introduces the target gene at the position where double-strand breakage occurs by the homology-directed repair (HDR, Fig. 7.2B) mechanism utilizing repair DNA as a template. In general, it is known that gene knock-in by the CRISPR/Cas9 technique that is required

(A)

Cas9

sgRNA

(B)

dCas

sgRNA

RNAP

Gene disruption

RNAP block RNAP

FIGURE 7.2 (A) Nonhomologous end joining (NHEJ), including proteins such as Ku repair double-strand breaks without a repair template. In the CRISPR/Cas9 technology, NHEJ results in variable length of indels. (B) Homologydirected repair (HDR) double-strand breaks by means of a repair template with homology arms. The knockout and knock-in can be performed precisely in the organisms whose HDR pathway is activated.

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for precise gene editing occurs at a low frequency, although the CRISPR/Cas9 technique allows gene knock-out at a high frequency (Chu et al., 2015). The knock-in and knock-out efficiency of the target gene of CRISPR/Cas9 by NHEJ and HDR can be influenced by the activity of the enzymes involved in the NHEJ and HDR machineries of the corresponding organisms. In the organisms where genes in the HDR pathway are more enhanced than NHEJ, the gene knock-in efficiency can be increased as compared to that of the organisms where the genes are suppressed. In addition to the gene knock-in problem, the off-target effect is repeatedly raised in studies using the CRISPR/Cas9 system. The offtarget effect is a problem in that guide RNA not only recognizes a 20-nucleotide sequence in the target gene but also nucleotide sequences in other positions in the genome. The incorrect recognition in the other sites of the genome can lead to unwanted or “off-target” breakage. This is a phenomenon that can occur more easily when the genome size is large or if a target sequence contains similar sequences in the genome. There is concern to overestimate an off-target effect of the CRISPR/Cas9 technique. However, it is necessary to estimate whether the mutations are caused by the CRISPR/ Cas9 technique through appropriate experimental control and statistical evaluation (Akcakaya et al., 2018; Willi et al., 2018). To overcome the limitation of the CRISPR/Cas9 technique, scientists have searched for better DNA nucleases possessing similar functions as Cas9. The Feng Zhang group discovered Cpf1 nuclease likely to be more precise and convenient than Cas9 nuclease (Zetsche et al., 2015). Later, Jin Su Kim’s group proved that the Cpf1 detected and cut the target sequence more precisely than Cas9, implementing the digenome-seq technique (Kim et al., 2016). In spite of the study claiming the superiority of the Cpf1 nuclease over Cas9, the CRISPR/Cas9 system has been preferentially used. While the application of the CRISPR/Cas9 technology into the metabolic engineering of the microbial cell factory has grown rapidly, the approaches to food microbes using the CRISPR/Cas9 technique are also noteworthy. Our group utilized CRISPR/Cas9 technology to decrease the production of carcinogenic chemical ethyl carbamate (EC) from the yeast strain Saccharomyces cerevisiae (Chin et al., 2016). EC is a carcinogen that potentially attacks human cells by mutating the Kras oncogene (Cazorla et al., 1998). The fermented foods, in particular alcoholic beverages, are prone to forming EC by reaction of ethanol and urea from yeast or other food microbes (Chin et al., 2016). In the study of our group, the key enzyme in the urea synthesis pathway was inactivated via the CRISPR/ Cas9 technology. The complete deletion of the target gene or inactivation induced by the nonsense mutation in the upstream of the target gene was executed by the CRISPR/Cas9 technique. The highly accurate and redundant alterations of the sequences on the target gene were conducted in the yeast system by the CRISPR/Cas9 technique, leading to the dramatic decrease of the precursor urea and EC (Chin et al., 2016). The Jay Keasling group also successfully employed the CRISPR/Cas9 technology to improve the fermentation process of alcoholic beverages (Denby et al., 2018). They introduced genes relevant to the “hoppy” flavor into a beer yeast strain. The introduction of the hoppy flavor appeared to simplify the beer-making process by eliminating the additional step of the addition of hops. As described before, the CRISPR/Cas9 technology has been utilized for and an influencer of various fields such as genetics, cell biology, gene therapy, food technology, etc. The CRISPR/Cas9 system on metabolic engineering, however, is one of the most important applications because the production of biopolymers through microbial cell factories appears primed to rely on the CRISPR/ Cas9 technique in the future.

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3. CRISPR/Cas9-based metabolic engineering Accurate genome editing is one of the main advantages of the CRISPR/Cas9 technique for metabolic engineering. The introduction of the nonsense mutation by the CRISPR technique induces the inactivation of the target gene to control and rewire metabolic circuits. Although the simple deletions of the genes involved in the competing pathways are the primary selection to decrease unwanted flow, the delicate control by modulating the expression levels could show an optimized result. This delicate control can be executed by modulating the levels of the gene employing the promoter containing the appropriate synthetic sequence. In addition, the desired phenotype obtained by global transcription machinery engineering (gTME) can be reintroduced into another host strain by altering the sequence of its homologous transcription factor to the desired sequence by the CRISPR/Cas9 technique. Although the delicate sequence change into the desired form has been accomplished by site-directed mutagenesis so far, the CRISPR/Cas9 technique is more convenient and amenable for implementation when high accuracy is needed. For example, some proteins enhance the fermentation performance of the host cell when the proteins are truncated (Kim et al., 2013). The precise truncation can be executed by the alteration of the target sequence on site of the genome through utilization of the CRISPR/Cas9 technique. The CRISPR/Cas9 technique can be used to induce multiple mutations on different genes, which usually require multiple markers and is inconvenient when conducted by conventional DNA manipulation. Since the coordination of multiple genes is important for the pathway optimization to acquire the desired phenotype of the host cells, the CRISPR/Cas system appears to broaden coverage in metabolic engineering. The usefulness of the coordination of the genes involved in various metabolic pathways by the CRISPR/Cas9 technique has been well documented in yeast systems. For example, the mevalonate pathway in budding yeast plays a critical role for production of the many important natural products such as isoprenoids (Asadollahi et al., 2010; Kirby et al., 2008; Verwaal et al., 2007; Engels et al., 2008). Multiple knock-in and knock-out of the genes involved in the mevalonate pathway is required for optimizing the productivity and yield for the desired products. Since limited selection markers can be used in yeast gene manipulation, recycling selection markers has been used for multiple gene coordination (Jakociunas et al., 2015). However, the recycling selection markers can cause unwanted chromosomal rearrangements by internal recombination between flanking homologous repeats (Solis-Escalante et al., 2014; Jakociunas et al., 2015). One of the important advantages of CRISPR/Cas9 is the ability to avoid unwanted chromosomal rearrangements. Corresponding to this, the Jay Keasling group successfully implemented CRISPR/Cas9 technology to modulate multiple genes involved in the mevalonate pathway in yeast to enhance the production of mevalonate, a key intermediate for the industrially important isoprenoid biosynthesis pathway (Jakociunas et al., 2015). They employed CRISPR/Cas9 technology for multiplex genome engineering of the knock-out of four target genes (BTS1, ROX1, YPL062W, and YJL064W) and the knock-down of one target gene (ERG9) to obtain a strain capable of highly producing mevalonate. In this study, when strain development was performed through all 31 possible target gene combinations, a strain with a 41-fold increase of mevalonate production, compared to the wild-type strain, was developed (Jakociunas et al., 2015). Similarly, Mans and his colleagues constructed the guide RNA vector for multiplex genome engineering using Gibson assembly for the CRISPR/Cas9-based approach (Mans et al., 2015). In addition to the multiplex genome engineering by gene knock-in and knock-out (Fig. 7.3A), CRISPR interference (CRISPRi, Fig. 7.3B) utilizing the nuclease-deactivated Cas9 (dCas9) is useful for metabolic engineering (Qi et al., 2013). CRISPRi by the dCas system can regulate genes involved in

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(A)

(B) Cas9

Cas9

crRNA

Single-guide RNA

tracrRNA FIGURE 7.3 (A) Normal CRISPR/Cas9 system. (B) CRISPR interference by dCas. RNA polymerase (RNAP) is blocked by the dCasesgRNA duplex, leading to the inhibition of the transcription.

the metabolic pathway transiently or constitutively to improve fermentation performance (Cho et al., 2018). The dCas9 is especially useful for the bacterial system because the normal Cas9 nuclease can confer programmed cell death to the bacterial strain by the breakage of the chromosomal DNA. The Cas9 DNA nuclease appears to be lethal to most bacterial species. The bacteria without appropriate DNA repair machinery, such as Ku and LigD, are unable to repair their chromosome from the doublestrand breakage of the chromosome (MacNeill, 2005). Therefore, the deactivated dCas9 interferes with the target genes by attaching to target sequences with guide RNA and can be efficiently exploited for engineering bacterial systems, such as E. coli, lacking adequate repair machinery. Another important advantage of the CRISPR/Cas9 system is the efficient and convenient gene knock-out in the diploid cells to generate a homozygous genotype. This function is critical when the gene is dominant and the loss of one allele of the gene has a negligible effect. In particular, it is difficult to generate a homozygous genotype in polyploidy cells possessing more than two complete sets of chromosomes, such as fungus and plant cells. The Yong-Su Jin group at the University of Illinois at Urbana-Champaign successfully implemented CRISPR/Cas9 technology to construct a quadruple auxotrophic mutant of an industrial polyploid S. cerevisiae strain (Zhang et al., 2014). They subsequently applied the CRISPR/Cas9 technique to the probiotic Saccharomyces boulardii that appeared to have a diploid nucleus (Liu et al., 2016; Hudson et al., 2014). They successfully developed a quadruple auxotrophic mutant of S. boulardii for further metabolic engineering. In the study of filamentous fungi such as Aspergillus sp., Christina Nødvig and her colleagues utilized the CRISPR/Cas9 system to mutagenize six species, of which one had not been genetically engineered before (Nodvig et al., 2015). Due to the difficulties of genetics in polyploid organisms, advances in research requiring genetic manipulation, such as metabolic engineering in eukaryotes, have been hampered. Moreover, if the breeding to generate the homozygous cells is challenging, the organism would be avoided as a host cell to produce the target molecule. Therefore, the advantage of the CRISPR/Cas9 technique in the genetic study of polyploidy cells can diversify options for selection of the host organism to generate desired products.

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4. CRISPR/Cas9-based genome editing in biopolymer production from prokaryotes Numerous bacteria have been harnessed for the production of biopolymers such as polylactic acid (PLA), polyhydroxyalkanoate (PHA), EPS, etc. The studies for PHA biosynthesis have been extensively studied so far to replace the need for the petroleum-based plastic. Many research groups have attempted to increase the metabolic flux toward PHA synthesis. The Guo-Qiang Chen group at Tsinghua University has successfully exploited the CRISPR/Cas9 system for PHA production from E. coli and the extremophile Halomonas spp. (Table 7.1) (Chen and Jiang, 2018). They regulated multiple essential genes in a PHA biosynthesis pathway using the CRISPRi system, allowing them to avoid the need for laborious and time-consuming multiple step gene manipulation (Lv et al., 2015). Repression of the genes in competing pathways by CRISPRi efficiently directed flux to the target monomer 4-hydroxybutyrate. They also engineered morphology of E. coli using CRISPRi to enhance PHA accumulation intracellularly. CRISPRi allowed the down-regulation of genes involved in the cell wall synthesis allowing the E. coli cell to become more elastic, creating more space for PHA accumulation (Zhang et al., 2018). In addition to engineering E. coli for enhanced production of PHA, the extremophile Halomonas spp., an industrially promising bacterial chassis, was harnessed to produce poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV), another common PHA (Tan et al., 2014; Chen et al., 2017). They repressed morphology-related gene ftsZ and PHA synthesis-related genes (gltA and prpC) in Halomonas spp. by CRISPRi to enhance the production of PHBV (Tao et al., 2017). In this study, the efficient repression by CRISPRi directed the flux to the competing pathways and balanced appropriate production of PHBV monomers. To obtain an optimized result, guide RNAs binding the different positions of the target genes were constructed and estimated the effect on the levels of the transcription of the target genes (Tao et al., 2017). They also were able to use the CRISPR/Cas9 system for the metabolic engineering of Halomonas sp. in further studies (Qin et al., 2018; Ling et al., 2018). As compared to the CRISPRi system, the CRISPR/Cas9 system can

Table 7.1 Biopolymers created by engineered microbes using CRISPR technology. Strains

Target products

CRISPR systems

References

E. coli

Poly(3-hydroxybutyrateco-3-hydroxyvalerate) Poly-3-hydroxybutyrate Poly(3-hydroxybutyrateco-3-hydroxyvalerate) Poly(3-hydroxybutyrateco-4-hydroxybutyrate) and poly(3hydroxybutyrate-co-3hydroxyvalerate) Glycan Hyaluronan Hyaluronan

CRISPRi

Lv et al. (2015)

CRISPRi CRISPRi

Zhang et al. (2018) Tao et al. (2017)

CRISPR/Cas9

Ling et al. (2018)

CRISPR/Cas9 CRISPRi CRISPRi

Ru¨tering et al. (2017) Westbrook et al. (2018a) Westbrook et al. (2018b)

E. coli Halomonas bluephagenesis H. bluephagenesis

P. polymyxa B. subtilis B. subtilis

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delete the desired genes permanently. While the constitutive deletion by the CRISPR/Cas9 system has various advantages in metabolic engineering, suitable expression level of a target enzyme by the CRISPRi system can be beneficial depending on the host cells. In addition to the intensive implementation of the CRISPR/Cas9 system in PHA production, CRISPR-Cas9-mediated genome engineering was conducted to diversify the EPS variants from Paenibacillus polymyxa (Ru¨tering et al., 2017). The CRISPR/Cas9-mediated gene deletion led to the alteration of monomer compositions and rheological features of EPSs from the mutants. EPSs are high-molecular-weight carbohydrate biopolymers possessing potential for application in medicine and the food industry, and other industrial purposes (Moscovici, 2015). Various bacteria, including lactic acid bacteria, are amenable for producing EPSs in suitable conditions (Zannini et al., 2016). In the food industry, EPSs from food microbes can determine rheological properties of foods (Zeidan et al., 2017). In addition, some EPSs appear to harbor biological functionality beneficial in biomedical, pharmaceutical, and cosmetic applications (Caggianiello et al., 2016). The Perry Chou group at the University of Waterloo implemented the CRISPRi system in Bacillus subtilis to enhance the production of the high-value biopolymer hyaluronic acid (HA) (Westbrook et al., 2018a). They controlled the membrane cardiolipin distribution by repressing the ftsZ gene, encoding a cell division initiator protein, via CRISPRi in order to improve the HA titer (Westbrook et al., 2018a). They also partially diverted the carbon flux from central metabolism into HA synthesis by reducing the expression of pfkA and zwf genes in the glycolytic and pentose phosphate pathways via CRISPRi, leading to a substantial improvement of the HA titer (Westbrook et al., 2018b). Although EPSs are valuable and promising, there is a lack of metabolic engineering studies for the production of EPSs from microbes. One of the possible reasons could be the involvement of numerous genes in EPS biosynthesis. Furthermore, it is a major hurdle to sequence the genome and to develop gene manipulation techniques individually for an EPS-producing microbe. Combined with recently evolved and increasingly cost-effective nextgeneration sequencing (NGS) techniques, the CRISPR/Cas9 system could be an excellent option for developing an engineered system to improve the EPS-producing microbes toward desired phenotypes (Fig. 7.4).

FIGURE 7.4 Metabolic engineering of the EPS-producing microbes using the CRISPR/Cas9 technology.

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Public concerns about genetically modified organism (GMO) safety impede the research of CRISPR/Cas9 techniques in food microbes. As the study of the CRISPR/Cas9 systems started originally with lactic acid bacteria, however, metabolic engineering of EPS-producing bacteria using endogenous CRISPR/Cas9 systems may be an attractive option. It is highly plausible, as many genomics studies focusing on EPS-producing bacteria have shown the presence of an endogenous CRISPR system in bacterial genomes (Wallace et al., 2014; Wu et al., 2014; Marcotte et al., 2017). Otherwise, it would be also possible to produce EPSs for food or medical purposes using a heterologous CRISPR/Cas9 system, followed by approval from regulatory bodies. This approach, however, can be time-consuming and complicated depending on government policy and consumer sentiment.

5. CRISPR/Cas9-based genome editing in biopolymer production from eukaryotes The metabolic engineering for lactic acid production in yeast can be applied to the usage as a monomer in PLA production (Ozaki et al., 2017). Mans and colleagues (Mans et al., 2017) attempted to identify transporters to export lactate in S. cerevisiae. This investigative study provided useful information for future metabolic engineering for lactate production through the deletion of putative lactate transporters utilizing the CRISPR/Cas9 technique (Mans et al., 2017). The Akihiko Kondo group at Kobe University successfully performed metabolic engineering of fission yeast via the CRISPR/Cas9 system to produce lactic acid from a glucose and cellobiose mixture (Ozaki et al., 2017). Using the CRISPR/ Cas9 system, they disrupted genes encoding two pyruvate decarboxylases: an L-lactate dehydrogenase, and a minor alcohol dehydrogenase to attenuate ethanol production. They also overexpressed acetylating acetaldehyde dehydrogenase and D-lactate dehydrogenase to increase the cellular supply of acetyl-CoA and D-lactic acid production (Ozaki et al., 2017). The engineered fission yeast efficiently produced D-lactic acid from both glucose culture and mixed culture with glucose and cellobiose. This study will provide useful information for the advancement of the PLA industry. There have been several efforts to produce PHA from S. cerevisiae (de Las Heras et al., 2016; Portugal-Nunes et al., 2017; Sandstrom et al., 2015). The budding yeast could be an alternative host to E. coli because of its own advantages as a workhorse over other microbial hosts: easy genetic manipulation, the simplicity of culture, food-grade status, posttranslational modification mechanisms, the presence of cellular compartmentation, etc. Introduction of the heterologous polyhydroxyalkanoate synthase gene allowed the production of PHA from S. cerevisiae (Portugal-Nunes et al., 2017). Presently, few studies, if any, employed the CRISPR/Cas9 system to engineer S. cerevisiae for PHA production, although the CRISPR/Cas9 system could be relatively easily implemented in S. cerevisiae to produce PHA. The utilization of the CRISPR/Cas9 system in S. cerevisiae is well established and DNA transformation is relatively easy as compared to other model organisms. The knock-out of the target genes can be efficiently performed via the CRISPR/Cas9 system. In the budding yeast system, however, efficiency of the knock-in of the target gene is also very high due to the HDR mechanism. Accurate genome editing is also very successful in S. cerevisiae. Therefore, the multiplex genome engineering for biopolymer production in budding yeast is possible through the CRISPR/Cas9 technique. In addition, the versatility of the CRISPR/Cas9 system can be applied to diverse fungal organisms as a biopolymer producer (Mahapatra and Banerjee, 2013). The endogenous synthesis pathways of

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biopolymers in numerous fungi can be employed. However, differences in the implementation of the CRISPR/Cas9 system are expected. Since the NHEJ pathway is enhanced in filamentous fungi, the gene knock-out via CRISPR/Cas9 appears to be operational. The precise genome editing in the filamentous fungi, however, may not as efficient as with budding yeast. By paying attention to several features of the fungal CRISPR/Cas9 system, a major impact on the production of biopolymers from fungi may be achieved.

6. Conclusions and perspectives Recent renovation in the scientific approach to genetic manipulation in microbes has been led by genome editing systems utilizing DNA nucleases such as zinc-finger nuclease, TALEN nuclease, and CRISPR/Cas. Among them, the CRISPR/Cas9 system is relatively convenient and widely applicable. The CRISPR/Cas9 technique is precisely editable and is capable of coordinating multiple genes. In addition, it is advantageous to manipulate the genome of polyploidy organisms, proving the CRISPR/Cas9 system can be an excellent genetic tool for eukaryotic genome manipulation. While numerous applications via the CRISPR/Cas9 system have been performed in all fields of life sciences, the CRISPR/Cas9 techniques have been heavily, and successfully, applied to microbial metabolic engineering. The multiplex genome editing via the CRISPR/Cas9 technique is suitable for the control of multiple genes involved in metabolic circuits. Not only knock-in and knock-out of target genes, but also repression of genes using CRISPR interference have had a profound impact in the metabolic engineering field. The CRISPRi systems are especially effective in organisms omitting genes necessary for DNA repair pathways. PHA and EPS production from prokaryotes has been facilitated by the CRISPR/Cas9 system, in particular CRISPRi. The CRISPR/Cas9 system has prosperously established itself not only in the conventional model organisms such as E. coli and B. subtilis but also in undomesticated organisms such as Halomonas sp. In eukaryotic microbes, the CRISPR/Cas9 technique has also been thoroughly implemented. Although few studies have illustrated the usage of the CRISPR/ Cas9 system for engineering eukaryotic microbes for the production of biopolymers, it appears to have great potential. The CRISPR/Cas9 system in budding yeast can be conveniently and efficiently executed to knock-in and knock-out target genes. Moreover, accurate genome editing is easily available in budding yeast via the CRISPR/Cas9 system. These benefits may eventually be utilized for the production of biopolymers in budding yeast. In addition to the benefits in the yeast CRISPR/ Cas9 system, the CRISPR/Cas9 technique may facilitate regulating genes in numerous pathways for biopolymer production in filamentous fungi. Collectively, the CRISPR/Cas9 system has already made a significant mark on the study and production of microbe-based biopolymers, and this impact is likely to rise in the coming years.

Acknowledgments This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07051143) and by the Ministry of Science and ICT (NRF-2018M3C1B5052439).

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