Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host

Cernak, Paul; Estrela, Raíssa; Poddar, Snigdha; Skerker, Jeffrey M.; Cheng, Yu-Ting; Carlson, Annika K.; Chen, Berling; Glynn, Victoria M.; Furlan, Monique; Ryan, Owen; Donnelly, Marie K.; Arkin, Adam P.; Taylor, John W.; Cate, J.H.D.

doi:10.1128/mbio.01410-18

Cited by 63 publications

(59 citation statements)

References 48 publications

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“…K. marxianus could be a suitable recombinant microorganism for the microbial production of pinene using whey permeate as a medium (Table 1). K. marxianus is a fast growing microorganism and has been observed to grow at temperatures greater than 50 • C [61][62][63]. K. marxianus has not traditionally been used in genetic engineering; however, K. marxianus' genome has been sequenced and tools have been developed for the genetic engineering of K. marxianus NBRC1777 [35].…”

Section: Whey As a Feedstock For Microbial Pinene Productionmentioning

confidence: 99%

“…K. marxianus has not traditionally been used in genetic engineering; however, K. marxianus' genome has been sequenced and tools have been developed for the genetic engineering of K. marxianus NBRC1777 [35]. A plasmid-based CRISPR-Cas9 was adapted for the genome editing of K. marxianus Km17 [63]. This enabled the engineering of stable heterothallic haploid strains of K. marxianus [63].…”

Section: Whey As a Feedstock For Microbial Pinene Productionmentioning

confidence: 99%

“…A plasmid-based CRISPR-Cas9 was adapted for the genome editing of K. marxianus Km17 [63]. This enabled the engineering of stable heterothallic haploid strains of K. marxianus [63]. Recombinant K. marxianus KM-L9-20 has been cultured in an optimized whey medium to overproduce lactase [34].…”

Section: Whey As a Feedstock For Microbial Pinene Productionmentioning

confidence: 99%

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The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

et al. 2020

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Pinene is a secondary plant metabolite that has functional properties as a flavor additive as well as potential cognitive health benefits. Although pinene is present in low concentrations in several plants, it is possible to engineer microorganisms to produce pinene. However, feedstock cost is currently limiting the industrial scale-up of microbial pinene production. One potential solution is to leverage waste streams such as whey permeate as an alternative to expensive feedstocks. Whey permeate is a sterile-filtered dairy effluent that contains 4.5% weight/weight lactose, and it must be processed or disposed of due its high biochemical oxygen demand, often at significant cost to the producer. Approximately 180 million m3 of whey is produced annually in the U.S., and only half of this quantity receives additional processing for the recovery of lactose. Given that organisms such as recombinant Escherichia coli grow on untreated whey permeate, there is an opportunity for dairy producers to microbially produce pinene and reduce the biological oxygen demand of whey permeate via microbial lactose consumption. The process would convert a waste stream into a valuable coproduct. This review examines the current approaches for microbial pinene production, and the suitability of whey permeate as a medium for microbial pinene production.

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Section: Whey As a Feedstock For Microbial Pinene Productionmentioning

confidence: 99%

Section: Whey As a Feedstock For Microbial Pinene Productionmentioning

confidence: 99%

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The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

et al. 2020

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“…In parallel, K. marxianus has been successfully used as a platform to produce heterologous compounds such as short-chain alcohols, carotenoids and polyketides (Cheon et al, 2014;Lin et al, 2017;McTaggart et al, 2019). However, all the latter studies focus on the overexpression of heterologous enzymes or pathways and only a few studies attempt engineer metabolism to improve production (Cernak et al, 2018;Kong et al, 2019). With regards to aromatic compounds, K. marxianus has been evolved to overproduce phenylalanine and by extension the aromatic alcohol 2-phenylethanol (2-PE) via an enhanced Ehrlich pathway (Kim et al, 2014), but this has shed little light on appropriate metabolic engineering strategies.…”

Section: Introductionmentioning

confidence: 99%

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

Rajkumar

Morrissey

2020

Preprint

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The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilize its metabolism as a basis for bio-based production. To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L−1 2-PE from minimal medium. The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.

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“…CRISPR technology opens avenues for genome editing and gene regulation (23,24). In sharp contrast to successful application in fungi, especially Saccharomyces cerevisiae (23,24), CRISPR editing in bacteria is problematic due mainly to the lack of corresponding DNA repair mechanisms (25). Derived from CRISPR-Cas9, CRISPR interference (CRIS-PRi) consists mainly of one or several subgenomic RNAs (sgRNAs) and a catalytically dead Cas9 protein (dCas9) lacking endonuclease activity (26).…”

mentioning

confidence: 99%

Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli

Zhao

et al. 2020

Antimicrob Agents Chemother

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Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Escherichia coli. Using CRISPRi to block plasmid R388 class 1 integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC50) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the dfrB2 cassette and sul1, two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of intI1, and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the intI1 nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in E. coli. This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR.

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Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host

Cited by 63 publications

References 48 publications

The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

The Potential Production of the Bioactive Compound Pinene Using Whey Permeate

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli

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