Design and Control of Extrachromosomal Elements in <i>Methylorubrum extorquens</i> AM1

Carrillo, Martina; Wagner, Marcel; Petit, Florian; Dransfeld, Amelie; Becker, Anke; Erb, Tobias J.

doi:10.1021/acssynbio.9b00220

Cited by 28 publications

(24 citation statements)

References 30 publications

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“…Moreover, cumate- or anhydrotetracycline-inducible hybrid promoters (P R/cmtO and P R/tetO ) with low basal expression and high expression upon induction have been developed ( Chubiz et al, 2013 ). Recently, Carrillo et al (2019) introduced a set of isopropyl β- d -1-thiogalactopyranoside (IPTG)-inducible promoters that were strong, tight, and controllable, even exceeding the promoter elements available thus far. However, IPTG is not suitable on an industrial scale because of its toxicity and cost ( Makrides, 1996 ; Browning et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Sathesh‐Prabu

Ryu

Lee

2021

Front. Bioeng. Biotechnol.

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Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/PhpdH) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%–100%) to that of the widely used strong promoters such as PmxaF and PL/O4. The HpdR/PhpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

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Section: Introductionmentioning

confidence: 99%

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Sathesh‐Prabu

Ryu

Lee

2021

Front. Bioeng. Biotechnol.

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“…Plasmid construction. The META1_1800 mutant allele was synthesized as a blunt-end 5'phosphrylated 1436 bp gBlock (IDT, Coralville, IA, USA) with the modified lac promoter PL/O4/A1 (Carrillo et al 2019) upstream. The gBlock was ligated into pCM66T digested with Ecl136II (Thermo Fisher Scientific, Waltham, MA, USA) to generate pNG327.…”

Section: Methodsmentioning

confidence: 99%

Harnessing methylotrophs as a bacterial platform to reduce adverse effects of the use of the heavy lanthanide gadolinium in magnetic resonance imaging

Good¹,

Er³

et al. 2021

Preprint

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Gadolinium is a key component of magnetic resonance imaging contrast agents that are critical tools for enhanced detection and diagnosis of tissue and vascular abnormalities. Untargeted post-injection deposition of gadolinium in vivo, and association with diseases like nephrogenic systemic fibrosis, has alerted regulatory agencies to re-evaluate their widespread use and generated calls for safer gadolinium-based contrast agents (GBCAs). Increasing anthropogenic gadolinium in surface water has also raised concerns of potential bioaccumulation in plants and animals. Methylotrophic bacteria can acquire, transport, store and use light lanthanides as part of a cofactor complex with pyrroloquinoline quinone (PQQ), an essential component of XoxF-type methanol dehydrogenases (MDHs), a critical enzyme for methylotrophic growth with methanol. We report robust gadolinium-dependent methanol growth of a genetic variant of Methylorubrum extorquens AM1, named evo-HLn, for evolved for heavy lanthanides. Genetic adaptation of evo-HLn resulted in increased xox1 promoter and XoxF MDH activities, transport and storage of Gd3+, and augmented biosynthesis of PQQ. Gadolinium-grown cells exhibited a shorter T1 relaxation time compared to cells with lanthanum or no lanthanide when analyzed by MRI. In addition, evo-HLn was able to grow on methanol using the GBCA Gd-DTPA as the sole gadolinium source, showing the potential of this strain for the development of novel GBCAs and gadolinium recovery from medical waste and/or wastewater.

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“…For further development of M. extorquens as an efficient platform in C1-biotechnology, a broad set of genetic tools is required. Several basic tools that have been developed for M. extorquens include versatile broad-host-range vectors based on a mutant IncP (Marx and Lidstrom 2001), cre-loxP-and sacB-based suicide vectors for allelic replacement (Marx and Lidstrom 2002), inducible promoters (Chubiz et al 2013;Kaczmarczyk et al 2013;Carrillo et al 2019), and transposon mutagenesis (Van Dien et al 2003b;Ochsner et al 2017). Yet, these genetic tools are far from complete, and many applications are challenged by the fact that more elaborate genetic tools for this bacterium are missing (Carrillo et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Several basic tools that have been developed for M. extorquens include versatile broad-host-range vectors based on a mutant IncP (Marx and Lidstrom 2001), cre-loxP-and sacB-based suicide vectors for allelic replacement (Marx and Lidstrom 2002), inducible promoters (Chubiz et al 2013;Kaczmarczyk et al 2013;Carrillo et al 2019), and transposon mutagenesis (Van Dien et al 2003b;Ochsner et al 2017). Yet, these genetic tools are far from complete, and many applications are challenged by the fact that more elaborate genetic tools for this bacterium are missing (Carrillo et al 2019). For the fine control of gene expression and metabolic flux, it is essential to develop a tool for efficiently knocking down gene expression in M. extorquens.…”

Section: Introductionmentioning

confidence: 99%

Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens

2021

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Aim: Genetic tools are a prerequisite for engineering cell factories for synthetic biology and biotechnology. Methylorubrum extorquens is an important platform for a future one-carbon (C1) bioeconomy, but its application is currently limited by the availability of genetic tools. Small regulatory RNA (sRNA) is an important regulatory factor in bacteria and has been applied for gene repression in several strains. This study aimed to construct a synthetic sRNA system based on the MicC scaffold and the chaperone Hfq to control gene expression in M. extorquens. Methods and Results: Initially, the exogenous lacZ gene was transposed into the M. extorquens chromosome as a reporter, and corresponding βgalactosidase was measured to assess the knockdown efficiency of lacZ. A synthetic sRNA containing a 24-nt antisense RNA targeting lacZ and an Escherichia coli MicC scaffold were constructed, and different Hfqs from E. coli, M. extorquens AM1 and PA1 were further identified. The results showed that the expression of endogenous hfqs from the chromosome in M. extorquens strains was inadequate, and only when it was overexpressed via the plasmid did the colonies show a colour change and a corresponding decrease in βgalactosidase expression. More specifically, M. extorquens strains with overexpressing their own Hfq showed the best gene repression efficiency. Furthermore, this E. coli MicC scaffold and AM1 Hfq system were combined to knock down crtI gene expression in AM1, leading to an 86% decrease in carotenoid production (0Á09 mg g −1 ) compared to that (0Á65 mg g −1 ) in the wild-type strain. Conclusion: A functional synthetic sRNA system combined with E. coli MicC and endogenous Hfq was constructed in M. extorquens strains, which was able to interfere with the target crtI gene and reduce carotenoid production. Significance and Impact of the Study: The synthetic sRNA system reported in this study provides a genetic tool for the manipulation of M. extorquens. The present findings might be helpful for achieving high-throughput gene knockdown expression.

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Design and Control of Extrachromosomal Elements in Methylorubrum extorquens AM1

Cited by 28 publications

References 30 publications

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens

Harnessing methylotrophs as a bacterial platform to reduce adverse effects of the use of the heavy lanthanide gadolinium in magnetic resonance imaging

Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens

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