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.
Highlights
It is is known known that that the the biosynthesis biosynthesis and and mis-incorporation mis-incorporation of of non-canonical branched-chain amino acids (ncBCAA) ncBCAAs into into recombirecombiIt nant proteins occurs during coli based recombinant protein production processes with nant proteins occurs during E. coli based recombinant protein production processes with perturbations in the glucose and dissolved oxygen
We postulate that the pulses with simultaneous limitation in a in fedthat the repeated repeatedapplication applicationofofpyruvate pyruvate pulses with simultaneous limitation a batch background triggers the the synthesis of ncBCAAs in ainsimilar way
As expected, its concentration was low at the end of the batch phase and only caused a very slight transient increase of formate, the pyruvate pulses together with coli BW25113 pSW3_lacI+ glucose limited fed-batch cultivation in a 15 L stirred tank reactor (STR)
Summary
The scale-up of recombinant protein production processes from laboratory scale to industrial scale reactors often leads to problems, which are basically connected to the longer mixing times in large-scale bioreactors. For an E. coli-based recombinant protein production process a 20% reduction of biomass yield and an increase of by-product formation was reported when scaling up from 3 L to 9 m3 [1]. It was reported that scaling up an E. coli process from 30 L to 450 L results in biomass reduction and a lower product yield [2]. Gradient zones of substrate, dissolved oxygen (DO), pH and other parameters are formed due to inefficient mixing and E. coli cells respond to these environmental changes by modulating their metabolism [3].
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