Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Abstract

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase^® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Keywords: mixed-acid fermentation; non-canonical branched chain amino acids; pyruvate pulse; scale-down; strain screening.

Figure 1

Measured OD₆₀₀ (a) and concentrations of recombinant mini-proinsulin (MPI) (b) over time after induction of E. coli BW25113 pSW3_lacI⁺ in a fed-batch cultivation in a 15 L bioreactor under reference cultivation conditions (blue symbols) or with pyruvate pulsing and concomitant dissolved oxygen (DO) limitation (red symbols). Results correspond to the average of three technical replicates. The first measured sample corresponds to the time-point right before induction.

Figure 2

Molar concentrations of norvaline (a), norleucine (b) and β-methylnorleucine (c) normalized to OD₆₀₀ in the intracellular soluble fraction after induction of E. coli BW25113 pSW3_lacI⁺ glucose limited fed-batch cultivation in a 15 L stirred tank reactor (STR) under reference cultivation conditions (blue symbols) or in a cultivation with pyruvate pulses and concomitant dissolved oxygen (DO) limitation (red symbols). Arrows indicate time points where 1 g L⁻¹ pyruvate pulse combined with 5 min DO limitation was applied. Results correspond to the average of 3 technical replicates.

Figure 3

Molar concentrations of norvaline (a,c) and norleucine (b,d) normalized to OD₆₀₀ (a,b) or to recombinant mini-proinsulin (MPI) mass (c,d) in the inclusion body fraction after induction of E. coli BW25113 pSW3_lacI⁺ glucose limited fed-batch cultivation in a 15 L stirred tank reactor (STR) under reference cultivation conditions (blue symbols) or in a cultivation with pyruvate pulses and concomitant DO limitation (red symbols). Arrows indicate time points of 1 g L⁻¹ pyruvate pulses combined with 5 min DO limitation. Results correspond to the average of 3 technical replicates.

Figure 4

Concentration of acetate (a) and formate (b) in culture broth supernatants during the feeding phase of a fed-batch cultivation E. coli BW25113 pSW3_lacI⁺ in a 15 L bioreactor under reference cultivation conditions (blue symbols) or under concomitant pyruvate pulsing and dissolved oxygen (DO) limitation (red symbols), respectively. Orange arrows indicate times of 1 g L⁻¹ pyruvate pulses each combined with 5 min DO limitation. Green arrow shows the time of IPTG induction. Results represent the average of 3 technical replicates.

Figure 5

Molar concentrations of norvaline (NV), norleucine (NL) and β-methylnorleucine (β-MNL) normalized to OD₆₀₀ in the hydrolyzed intracellular soluble protein fraction (a) and inclusion body fraction (b) at 3 h after induction of E. coli BW25113 pSW3_lacI⁺ cultivation in 10 mL mini-bioreactor cultivations under reference conditions (Reference) or under simultaneous pyruvate pulsing and dissolved oxygen (DO) limitation (Pyr pulses and DO lim.). Results represent the average of 3 independent biological replicates.