A plug-and-play system for enzyme production at commercially viable levels in fed-batch cultures of Escherichia coli BL21 (DE3)

Abstract

Commercial exploitation of enzymes in biotransformation necessitates a robust method for enzyme production that yields high enzyme titer. Nitrilases are a family of hydrolases that can transform nitriles to enantiopure carboxylic acids, which are important pharmaceutical intermediates. Here, we report a fed-batch method that uses a defined medium and involves growth under carbon limiting conditions using DO-stat feeding approach combined with an optimized post-induction strategy, yielding high cell densities and maximum levels of active and soluble enzyme. This strategy affords strict control of nutrient feeding and growth rates, and ensures sustained protein synthesis over a longer period. The method was optimized for highest titer of nitrilase reported so far (247 kU/l) using recombinant E. coli expressing the Alcaligenes sp. ECU0401 nitrilase. The fed-batch protocol presented here can also be employed as template to produce a wide variety of enzymes with minimal modification, as demonstrated for alcohol dehydrogenase and formate dehydrogenase.

Dynamic flux balance analysis of high cell density fed‐batch culture of Escherichia coli BL21 (DE3) with mass spectrometry‐based spent media analysis

“…The starter culture was grown overnight at 37°C and 180 rpm in LB medium containing 0.15 mg/mL carbenicillin. The production culture was initiated with 100 mL working volume and initial OD 600 of 0.05 (Sohoni et al, 2018) in a 250 mL autoclavable minibioreactor (Applikon). Super optimal broth (SOB) supplemented with 30 mM glycerol, 10 mM MgCl 2 , and 0.15 mg/mL carbenicillin was used as the initial medium (Sohoni et al, 2015), while the feed medium contained 17.5% w/v glycerol, 24.5% w/v yeast extract, 2 mM MgCl 2 , and 0.3 mg/mL carbenicillin (Ganjave et al, 2022).…”

Precision fermentation with mass spectrometry‐based spent media analysis

“…The development of an efficient bioprocess requires the control and tuning of several process parameters, including pH, temperature, media type and composition, rate of agitation, gas flow rate, and nutrient feeding. Such macrolevel optimization is commonly achieved using experimental design strategies like Plackett–Burmann and response surface methodologies, and controlled feeding techniques such as dissolved oxygen (DO) stat, pH‐stat, and Hi‐end pH delivery of glucose (HiPDOG) (Gagnon et al, 2011; Sohoni et al, 2015, 2018). Other strategies involve the addition of specific salts or metabolites, which improve cell growth and recombinant protein yield, albeit through affecting the cell physiology rather than specifically targeting the protein or metabolite of interest (Datta et al, 2013).…”