3.2.1 Genetic escape variants of GFP-producing derivatives
Out of the 24 BQ<A1-GFP> derivatives isolated after directed evolution, the genomes of two randomly selected derivatives were sequenced, since all of them exhibited the same phenotype after six passages. Interestingly, both derivatives exhibited exactly the same mutation within the lacIQpromoter (Table 1). Next to the introduced C→T substitution[25] within the -35 region of the LacIpromoter, we found a G→A transition. However, this mutation has no influence on the basal expression or tunability of the A1 expression system (Figure S3).
Of the 24 B3<T7-GFP> derivatives, we sequenced the genomes of the 11 isolated subclones (Figure 1C). The mutations found in the B3<T7-GFP> derivatives can be divided into three clusters: mutations within the T7 promoter or T7 RNAP gene, mutations within genes involved in the metabolism and transport of various sugars, and mutations within the lac operon. Among the non-producing and weakly producing strains (subclones S1-S4, Figure 1C), mutations within the T7 RNAP gene or the T7 promoter were responsible for the observed phenotype. Derivative #E7P6 S1 had a 13-bp deletion within the T7 promoter encompassing almost the complete T7 promoter sequence. In #A5P6 S1, 10,872 bp of the DE3 lysogen were deleted, harboring the complete sequence of the T7 RNAP gene. In general, no mutational hotspot within the T7 RNAP gene was seen. In each sequence of the B3<T7-GFP> derivatives, which had a mutation in the T7 RNAP, we found a different SNP or indel that caused a loss of function of the T7 RNAP.
As a second cluster, we identified mutations within genes involved in the metabolism and transport of various sugars. One mutation found in the A1 and T7 expression system was a C→T mutation within thegntT operator site.[42, 43] The influence of the gluconate transporter on lactose-inducible gene regulation and its influence on growth during production cannot be fully explained because the medium used for cultivation did not contain any gluconate. Derivative #B8P6 S2 exhibits a T → G transversion within the promoter sequence of setA . SetA is an efflux pump capable of transporting a range of sugars and sugar analogues. Cells overexpressing SetA exhibit decreased accumulation of lactose and IPTG.[44] We speculate that this mutation increases the SetA expression rate, preventing intracellular accumulation of IPTG and reducing the induction of the GOI.
Derivative #A5P6 S5 has a T→C mutation within ptsI , leading to a L256P substitution. PtsI is a cytoplasmic protein that serves as the gateway for the phosphoenolpyruvate:sugar phosphotransferase system (PTSsugar). E. coli mutants with < 1% residual PtsI activity are unable to consume the PTS sugars glucose, fructose, mannose, mannitol, sorbitol, N-acetylglucosamine, and N-acetylmannosamine, and the non-PTS sugars glycerol, melibiose, maltose, and lactose [45]. The L256P substitution may reduce PtsI activity to a level at which glucose can still be used as a substrate, but the intake of IPTG is reduced, which also reduces induction of the GOI.
In addition to a mutation within the T7 RNAP gene, derivative #A5P6 S7 also exhibited a mutation within malP . The (T)54mutation causes a frameshift and probably complete loss of function. MalP is involved in maltose metabolism and has high affinity for short, linear α-1,4 linked oligoglucosides.[46] A mutation within srlR was found in derivative #B8P6 S8. This gene encodes the glucitol repressor GutR and is a DNA-binding transcription factor that represses the gut operon involved in the transport and utilization of glucitol.[47]
A mutation in the type III CoA transferase gene, caiB[48], was found in four derivatives. It is unlikely that the mutation in caiB influences recombinant protein production or growth behavior, because all B8 derivatives, which can be classified as both strong and weak producers, exhibit this mutation.
Mutations in the third cluster comprise the lac operon itself. Two mutations were found in the pLac promoter in derivatives belonging to strongly producing subclones S9 and S10. The positive properties of mutations in pLac on lac -regulated recombinant gene expression are already known [19]; expression of thelac operon is reduced, including reduced expression of LacY, the sugar transport protein. Consequently, less IPTG enters the cell, which weakens induction of the GOI.[49] The metabolic burden is reduced, explaining why the selected derivatives no longer have a disadvantage in growth. We speculate that this phenomenon also applies to the mutations observed in other genes, such as setA ,ptsI , malP , srlR, and gntT, which are involved in the metabolism of various sugars, even though they are not known to be directly related to IPTG or glucose transport into the cell. However, a negative influence on recombinant protein production can only be clearly attributed in the case of ptsI , as this mutation does not occur in combination with a mutation in the T7 promoter or in the T7 RNAP gene. Unfortunately, this is not the case with mutations insetA and malP . However, the mutations in srlR andgntT clearly have a positive influence on the growth rate under conditions of recombinant protein production.
Table 1. Mutations in GFP-producing derivatives of B3<T7-GFP> and BQ<A1-GFP> isolated after fractionation.