Methods and Systems for Methylotrophic Production of Organic Compounds

Example 1

Optimization of Growth Medium for Paracoccus sp. when Using Formate as the C1 Compound

[0257]Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 were obtained from the American Type Culture Collection (ATCC).

[0258]Strains were tested for the ability to grow aerobically on sodium formate as a sole source of carbon and/or energy using MOPS minimal medium (Teknova, Inc.) with sodium formate as a sole carbon source at 37C. Unlike previous media used to evaluate the formate-dependent growth of Paracoccus, this medium contains defined levels of trace elements molybdenum, boron, copper, zinc, manganese, and other trace metals.

[0259]Growth was conducted in various high-throughput machinery capable of monitoring growth by light scattering at 600 nm, including a Gemini SpectraNax plate reader (Molecular Devices, Inc.), a Tecan M3000 plate reader (Tecan, Inc.), and a BioLector device (m2p-labs, Inc.). For the BioLector, the CO2 gas content in the culture headspace was controllable, as was the humidity.

[0260]Paracoccus zeaxanthinifaciens ATCC 21588 was incapable of growth on formate as a sole carbon or energy source. The other two Paracoccus strains were capable of growth on formate as a sole carbon source, as has been previously reported [Microbiology, 1979, 114(1):1-13, DOI: 10.1099/00221287-114-1-1; Arch Microbiol, 1978, 118(1):21-26, DOI: 10.1007/BF00406069].

[0261]The effect on growth rate of changes in temperature (T, in degrees Celsius), the partial pressure of CO2 in the culture headspace gas (pCO2, in percent by volume), shaking speed (in rpm), and/or the concentrations (in mM) of sodium formate (HCOONa), sodium nitrate (NaNO3), sodium thiosulfate (Na2S2O3), sodium chloride (NaCl), and sodium bicarbonate (NAHCO3) added to the Basal MOPS minimal medium were systematically evaluated. Combinations examined are shown in Table 2. Paracoccus strains are labelled according to their ATCC number. For each measurement, the instrument used (BioLector; SpectraMax; Tecan), plate type (Flower, Flower Plate; 96 transp, 96-well transparent microtiter plate; 96 opaque, 96-well opaque microtiter plate), use of plate lid, use of humidity control and total culture volume in uL is indicated. N/A indicates that a particular experimental condition is not applicable for the instrument used.

TABLE 2 Tested growth conditions for each Paracoccus strain Shaking Humidity ATCC NaHCO3 NaCl NaNO3 Na2S2O3 HCOONa T speed Instrument Plate Lid Control pCO2 Vol 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 37.0 1200 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 150.0 0.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 60.0 0.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 60.0 0.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 80.0 50.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 13534 0.0 0.0 0.0 0.0 20.0 34.0 900 BioLector Flower N/A TRUE 5% 1300 25364 10.7 4.3 4.3 40.0 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 13534 75.0 4.3 4.3 5.7 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 13534 10.7 4.3 30.0 5.7 45.7 36.6 1157 BioLector Flower N/A TRUE 5% 1300 25364 42.9 17.1 17.1 0.0 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534 0.0 17.1 17.1 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 13534 42.9 17.1 0.0 22.9 32.9 35.3 1029 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 11.4 26.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 25364 101.0 6.1 6.1 0.0 26.7 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.7 16.3 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 13534 122.4 29.4 14.7 0.0 67.8 34.4 937 BioLector Flower N/A TRUE 5% 1300 21588 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 unknown Tecan 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 N/A SpectraMax 96 transp TRUE N/A N/A 150 21588 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 31.0 Linear (8.5) Tecan 96 transp TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque TRUE FALSE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 Linear (8.5) Tecan 96 opaque TRUE N/A N/A 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 800 BioLector 96 opaque TRUE TRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 150 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 100 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 150 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector 96 opaque FALSE TRUE FALSE 100 25364 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE FALSE 1300 13534 0.0 0.0 0.0 0.0 50.0 37.0 1200 BioLector Flower N/A TRUE FALSE 1300

[0262]The particular values for salt, bicarbonate, formate, thiosulfate, or nitrate concentration, as well as temperature, were chosen by implementing a Nelder-Mead simplex optimization algorithm (as described in Chapter 18 of Chemometrics: a textbook ISBN: 0444426604) using the starting simplices with points chosen from the following possibilities: temperature, 34° C. or 37° C.; sodium bicarbonate, 150 mM or 0 mM; sodium chloride, 60 mM or 0 mM; sodium formate, 20 mM or 50 mM; sodium nitrate, 60 mM or 0 mM; sodium thiosulfate, 0 mM or 80 mM; shaking speed, 1200 rpm or 900 rpm. Growth was evaluated for both 25364 and 13534 at each chosen medium condition. For each strain and medium condition, a score indicative of the growth was calculated as the time (in hours) to 50% of the maximum growth attained in the entire experiment minus the time to 5% of the maximum growth. This metric is easy to compute and avoids penalizing conditions with longer lag phases.

[0263]From the scores, new medium conditions were calculated according to the Nelder-Mead simplex algorithm. These medium conditions were tested as well. The growth under the new condition as well as the old ones was used to define the points of a new simplex, and the process repeated.

[0264]After several rounds of the medium optimization process, a satisfactory medium condition, allowing for faster growth than the initially chosen medium conditions, was obtained. In total, 68 different unique medium/strain/temperature/shaking conditions were examined. The fastest growth on formate as a sole carbon source was obtained for ATCC strain 25364. Under the optimal conditions, the medium consisted of 100 mM sodium bicarbonate, 6 mM sodium chloride, 6 mM sodium nitrate, 11 mM sodium thio sulfate, and 26 mM sodium formate in addition to standard MOPS minimal medium components. The optimal growth temperature was 34° C. Under these conditions, ATCC strain 25364 had a growth rate of >0.7 hr−1, corresponding to a doubling time of 0.95 hr.

[0265]ATCC strain 25364 was found capable of growth at rates in excess of 0.4 hr−1 using a simpler medium composition of MOPS minimal medium plus 50 mM sodium formate.

[0266]Large concentrations of thiosulfate were found to give slowed, biphasic growth curves for both strains of Paracoccus tested. In general, moderate concentrations of sodium nitrate showed improved growth. Growth at 34° C. was slightly better than growth at 37° C., and both of these temperatures were significantly better than growth at 30° C.

Example 2

Automatable Protocol for Conjugative Transfer of Plasmids from E. coli Donors to Paracoccus sp

[0267]E. coli strain 517-1 was obtained from the Yale E. coli Genetic Stock Center. Paracoccus denarificans PD1222 was obtained from Stephen Spiro (University of Texas at Dallas). E. coli S17-1 strain is tra+, meaning it is able to mobilize for conjugative transfer those plasmids harboring a mob+genotype. Plasmid pDIY313K, obtained from Dariusz Bartosik (University of Warsaw, Poland) and described by his laboratory [J Microbiol Methods, 2011, 86(2):166-74, DOI: 10.1016/j.mimet.2011.04.016], and introduced into E. coli 517-1 by standard methods.

[0268]E. coli S17-1 was grown on Luria broth with carbenicillin overnight. Paracoccus versutus ATCC 25364 was grown overnight on MOPS minimal medium (Teknova, Inc.) with 50 mM sucrose and 40 mM sodium nitrate.

[0269]The next day, E. coli S17-1 was subcultured in antibiotic-free Luria broth for >4 hr. Paracoccus strains were subcultured in identical MOPS/sucrose/nitrate medium. After cultures of both E. coli and Paracoccus had reach log phase, with optimal density greater than 1.0 cm−1, cultures were mixed in equal volumes in wells of a standard, SBS-format 96-well plate. No effort was made to pellet the cells, to immobilize cells on porous filters, to culture the cells on solid media, or to otherwise manipulate the mixtures. Cultures were simply mixed in equal volumes and incubated overnight at 37° C. without agitation.

[0270]After overnight incubation, the mixed cultures were diluted in PBS and dilutions were plated on MOPS/sucrose/kanamycin agar. E. coli cannot use sucrose as a carbon source, and only strains carrying pDIY313K can grow in the presence of kanamycin. Thus on these plates only transconjugants—strains of Paracoccus containing plasmid DNA and expressing plasmid-derived kanamycin resistance genes—can grow. In parallel we plated the same dilutions on MOPS/sucrose agar without kanamycin, in order to calculate the cell concentration of total Paracoccus cells used in the experiment and to calculate the transconjugation frequency (colonies of plasmid-bearing Paracoccus isolated per colony of recipient Paracoccus cell).

[0271]Using this simple technique we were able to demonstrate conjugation frequencies of 10−5 using Paracoccus denitrificans PD1222 and 2×10−7 using Paracoccus versutus. It should be emphasized that this frequency was determined via a protocol which did not require non-selective growth on soft medium, the use of filters, or any centrifugation steps. These steps are required in protocols for conjugation frequently taught in the literature. For example, Bartosik [J Microbiol Methods, 2011, 86(2):166-74, DOI: 10.1016/j.mimet.2011.04.016] teaches that cells must be grown, pelleted by centrifugation, washed, resuspendend, mixed, immobilized on a porous filter, grown under non-selective agar overnight, removed from the filter by washing, pelleted, and finally plated on selective medium. The lack of any such laborious cell manipulation procedures in our protocol is essential for conduction of the protocol on a robotics-based liquid-handling platform, where centrifugation and resuspension operations are much more error-prone, hard to implement, and/or unreliable in comparison with simple liquid handling steps.

Example 3

Genome Sequencing of Paracoccus Strains

[0272]Genomic DNA was isolated from Paracoccus zeaxanthinifaciens ATCC 21588, Paracoccus versutus ATCC 25364, and Paracoccus denitrificans ATCC 13534 using a Wizard Genomic DNA Isolation Kit (Promega, Inc.). The resulting DNA samples were fragmented and converted to paired-end libraries for whole-genome shotgun sequencing on a 454 pyrosequencing platform (Roche, Inc.).

[0273]For Paracoccus denitrificans, 37,585,886 paired reads, each 100 nt in length, were obtained. This represents approximately 3.7 gigabases of sequence data, or approximately 730-fold coverage of the 5.2 megabase genome of Paracoccus denitrificans PD1222 (Genbank accession numbers CP000489, CP000490, and CP000491 for chromosome 1, chromosome 2, and a 653,815 bp megaplasmid, respectively). Reads were assembled first by de-novo assembly and second by mapping the de-novo contigs to the published PD1222 genome.

[0274]The resulting reads could be assembled into a crude whole-genome assembly of 351 scaffolds comprising 21,972,742 total reads. The maximum scaffold was 7974 nt and minimum-length scaffold 2004 nt.