Abstract
Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. Methylorubrum extorquens AM1 is an important platform organism of a future C1-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested repABC regions to maintain extrachromosomal DNA in M. extorquens. We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between Escherichia coli and M. extorquens. These mini-chromosomes are compatible among each other and with high-copy number plasmids of M. extorquens. We also developed a set of inducible promoters of wide expression range, reaching levels exceeding those currently available, notably the PmxaF-promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in M. extorquens, which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.
Highlights
We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between Escherichia coli and M. extorquens
A single point mutation short, nontranslated counter-transcribed RNA typically located in the repB/repC intergenic region.[15−17] On within the O4s region of PA1/O4s, resulting in PA1/O4s_GA, showed increased mCherry expression at the expense of the basis of this distinctive region, a new family of repABC-type shuttle vectors was successfully designed for Sinorhizobium meliloti mimicking the characteristic features of secondary replicons, i.e., single copy number and stable propagation.[18]
We provide a set of novel promoters for M. extorquens AM1 that are tight and show a dynamic range of different expression levels upon induction, even exceeding the promoter elements available far
Summary
Realization of Tight, IPTG-Inducible Promoters with a megaplasmids. The replication of megaplasmids is integrated Dynamic Range. The replicons were isolated from kanamycin resistant cells after 96 h without selective pressure and their integrity was verified by restriction analysis (data not shown) These three replicons allowed fast doubling times of M. extorquens, reaching almost wildtype-like growth behavior, and were present at a copy number of 1 (Table 1), indicating that they are good candidates for the construction of mini-chromosomes. We decided to assemble several mini-chromosomes from the four basic modules, i.e., (1) a repABC-based origin for M. extorquens AM1 (Mex-DM4, Nham-3, Mrad-JCM, MexCM4), (2) an E. coli origin of replication (pMB1, p15A, or pSC101*), (3) a minimal antibiotic resistance cassette (Kanamycin, Gentamicin, Tetracycline), and (4) a multiple cloning site (MCS1, MCS2, MCS3) flanked by synthetic terminators (Table 2). Double transformations were possible, adding to the convenience of these tools for their use in genetic engineering of M. extorquens AM1
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