One-pot assembly of a model reporter chemicals production plasmid and a stabilised variant
Type IIS restriction assembly techniques were used to assemble two variant plasmids for citramalate expression and reporting. This allowed the exact order of the operon components to be arranged specifically and also assembled all in a ‘one-pot’ process. Figure 2 shows the components and products of this assembly. A pET29a(+) backbone was chosen as a widely used expression cassette, from which a 3573bp sized fragment of the backbone containing the rop and kanamycin resistance (kanr) genes, as well as the pBR322 origin, was cloned by PCR and purified by gel electrophoresis. The fresnoRFP gene was assembled into a plasmid alongside the cimA3.7 gene to generate a reporter linked to a model chemicals production pathway, both of which were expressed under the Anderson collection constitutive promotor BBa_J23119 (Kelly et al., 2009). This generated the plasmid pQR2401, described in Figure 2. The cer fragment was included as a further DNA part in the assembly upstream of the production-reporter operon to generate plasmid pQR2402, as described in Figure 2. This arrangement allows for any combination of pathway genes and/or reporters to be assembled using the same method, generating a potential tool for the easy generation of industrially relevant strains in the future.
Retention of expression throughout continuous fermentation
BW25113 ΔldhA cells were transformed with either of the resultant plasmids pQR2401 or pQR2402, the same host strain described previously as demonstrating high levels of product formation(Webb et al., 2018). Cells were then grown in a glucose-limited chemostat for 106 hours, in defined media, without selective pressure to determine the length of production of the chemical citramalate, determined by HPLC.
Glucose concentration in the chemostat dropped for the first 12 hours until steady state was reached, and the reactor became carbon-limited. Glucose levels then remained constant until the end of the fermentation. Citramalate production from cells containing plasmid pQR2401 climbed sharply to a maximum of 2.39 g L-1 at 12 hours, began to drop immediately after this high point and decreased to half of the maximum level by 33 hours. This indicates that there was no stable citramalate production period without any additional plasmid stabilisation.
When the cer fragment was added to the plasmid (pQR2402), peak citramalate production was detected at the same time point, 12 hours, and at a lower amount, 1.92 g L-1. However, production was retained around maximum levels for 58 hours before a drop in production was detected, reaching half this maximal level at 74 hours. Total citramalate production can be inferred from the area underneath the concentration plot (Table 1). Despite the initially higher citramalate production, the increased stability provided by the cer fragment resulted in nearly 1.6 times the amount of chemicals production over the fermentation lifetime.
The total citramalate production determined from the area underneath the concentration curve over the course of the 106-hour continuous fermentation.
||Total citramalate production (g)
||Bulk fluorescence detected (AU)
Due to the operonic nature of the plasmid assembly, fluorescence is initially linked to expression of the citramalate gene. As a result, monitoring the fluorescence signal from samples taken during the fermentation allows plasmid expression and retention to be tracked by simple excitation/emission studies, simple spectroscopic analyses that are quicker, cheaper, and easier to scale than HPLC (Rodrigues et al., 2018)(Kiviharju et al., 2007). Fluorescence readings can be performed in seconds, on pure samples, without the need for processing steps. Fluorescence values of samples from cells containing pQR2401 and cells containing pQR2402 both peaked at 20 hours with cells containing pQR2401 dropping to half maximal fluorescence at 41 hours and cells containing pQR2402 by 77 hours. The delay compared to citramalate production may reflect maturation time delays required for fluorescence proteins(Hebisch et al., 2013). Maturation time and stability can vary and the manufacturer does not provide this information, however in general, maturation for red fluorescent proteins has been shown to vary between approximately 0.5h and 3h (Balleza et al., 2018). Lysed cells may also release fluorescent proteins into the media which may be retained and thus give an extended reading for retention that does not accurately match productivity time. RFP is a useful indication of absolute gene retention and pathway expression levels, however, when measured as a bulk value it does not give a complete or accurate picture of how expression of pathway genes is affected over time. Spectroscopic analysis can, however, provide a more comprehensive understanding of phenomena that cannot be identified with conventional tools.
Single-cell measurements of plasmid retention
The use of flow cytometry allows for single-cell level analysis giving more information than is available from bulk fluorescence or chemical concentration values alone. The ability to measure fluorescence levels of individual cells enables tracking of single-cell gene expression. The measurement of any fluorescent signal within a cell above a control value demonstrates retention of pathway expression (therefore no total plasmid loss and no global operon structural changes) and the value of the fluorescence of each cell demonstrates the expression levels of the operon at a single cell level.
When no additional stabilisation is present on the production plasmid, the percentage of cells retaining an RFP signal above control begins dropping after only 12 hours (Figure 4a, blue line), matching the observed citramalate production more closely than bulk fluorescence values. Cells seem to retain plasmid and expression during the batch phase, but non-expressing cells are accumulated as soon as the fermentation enters a carbon-limiting state. Addition of cer to the plasmid prevents accumulation of non-expressing variants arising until after 58 hours (Figure 4a, red line). This matches the profile of citramalate production showing that the single-cell fluorescence analysis is an excellent proxy for retention of plasmid-borne production genes. Half of the population no longer demonstrates RFP fluorescence by 45 hours without stabilisation, increasing to 71 hours when cer is included on the expression plasmid (Table 2).
The calculated half-lives based on a regression fit of the RFP+ percentage retention data, the time at which half of the cells no longer exhibit any fluorescence expression and are therefore likely to have lost plasmid, in a continuous fermentation at a dilution rate of 0.1 h-1. The overall productivity across the entire fermentation and the peak productivity at the maximal citramalate productive period are shown as grams of product produced, per litre of fermentation broth, per hour. The product yield is shown as the amount of citramalate produced compared to the glucose added throughout the length of the fermentation.
|Half life (hours)
|Overall Productivity (g.L-1.h-1)
|Peak Productivity (g.L-1.h-1)
|Product Yield (gcitramalate.gglucose-1)
The median expression level of cells that retain an RFP signal above control increases throughout the continuous fermentation of pQR2401 containing cells, showing an increase of approximately four-fold from the steady state levels at 20 hours to the maximum at 90 hours (Figure 4b). Although the proportion of cells retaining fluorescent protein expression is decreasing, those that still contain plasmids have an increased total expression level. In addition, the variance of this population increases in a similar way showing that the expression levels become variable as the fermentation progresses (Figure 4c). It is likely that plasmids are being unevenly segregated at cell division, leading to a wide array of different copy numbers in the population, leading to a wide variety of pathway expression. In contrast, addition of the cer fragment to the expression plasmid results in a comparatively stable median fluorescence that peaks two-fold higher at 82 hours than at 20 hours, which demonstrates that cer has increased the likelihood of even plasmid segregation at cell division. Variation is lower throughout when cer is included in the expression plasmid, showing that expression levels are more consistent across the population. Median fluorescence does fall sharply when cer is present after 82 hours, showing that pathway expression is still lost. It is possible that by this point in the fermentation structural instability may have become limiting.
The eventual loss of fluorescence in cer-containing cells suggests that cer does not guarantee high copy-number maintenance indefinitely but that it does decrease the likelihood of plasmid loss at each cell division. Eventually, loss of productivity does still occur, and non-productive plasmid free cells do arise. Further studies, potentially utilising next generation genomics techniques, will be needed to discover what is causing the loss of productivity when segregational stability has been increased with cer addition.
Peak productivity (i.e. the productivity when maximal citramalate production was achieved) was higher for cells lacking cer on the production plasmid, supported by the higher median fluorescence of cer- cells at this time. Inclusion of cer appears to stabilise the plasmid copy number, allowing for greater stability with a small reduction in maximial productivity. The overall productivity, however, was 60% higher when cer was included in the plasmid (Table 2). This reflects the trend seen in the concentration profiles (Figure 3a and b) and demonstrates that without the addition of cer, cells lose productivity from their plasmids earlier in the continuous fermentation. This had a similar affect on the yield of the process, with cer+ cells demonstrating 8% higher conversion of glucose to the citramalate product.