Glucose Limited Feed Strategy Leads to Increased Production of Fusicocca-2
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Halka et al. AMB Expr (2018) 8:132 https://doi.org/10.1186/s13568-018-0662-8 ORIGINAL ARTICLE Open Access Glucose limited feed strategy leads to increased production of fusicocca‑2,10(14)‑diene by Saccharomyces cerevisiae Lisa Marie Halka1, Christian Nowacki2, Alica Kleinschmidt1, Kevin Koenen1 and Rolf Wichmann1* Abstract Fusicocca-2,10(14)-diene (FCdiene) is a diterpene which is interesting as a precursor of the anti-cancer drug fusicoccin A and therefore for pharmaceutical applications. Production of FCdiene using a genetically modifed Saccharomyces cerevisiae has been previously demonstrated with batch cultivations with a product concentration up to 10 mg/L. However, it is widely known that fed-batch processes can signifcantly improve product titer in yeast fermentations. This study focuses on the establishment of fed-batch fermentation for FCdiene production because fed-batch cultivations ® using FeedBeads indicated that limiting glucose supply could increase yields of biomass (1.07 gCDW/gGlucose instead of 0.20 gCDW/gGlucose) and FCdiene (21.54 mgFCdiene/gGlucose instead of 9.74 mgFCdiene/gGlucose) in shake fask scale and may have implications for larger scale processes. We implemented a new exponential glucose feed profle in a 1.8 L stirred tank reactor. This reduced overfeeding and the consequent, ethanol production. As a result improvements in cell concentrations up to 246% could be achieved and FCdiene yield increased up to 2.8X in the frst 28 h. FCdiene concentration reached 161 mg/L and 320 mg/L at 44 h. Fed-batch and batch mode were combined to examine dynamics of bi-modal cultivation where a fed-batch phase was used for biomass production and a batch phase used for FCdiene production potentially supported by ethanol consumption as reported on production of betulinic acid. The present study highlights the potential of process development improvements which increase high-value heterologous diterpene yields from S. cerevisiae. Keywords: Fusicocca-2,10(14)-diene, Fed-batch fermentation, Feed strategy, Model of feed control, Production improvement, Potential of process development Introduction (IPP) and its isomer dimethylallyl pyrophosphate Te tricyclic fusicocca-2,10(14)-diene, also known as (DMAPP) (Breitmaier 2005; Ruzicka 1953). fusicoccadiene (FCdiene), belongs to the family of dit- Fusicoccanes have recently gained attention as interest- erpenes and is one of the fusicoccanes (Muromtsev ing compounds for their apoptotic and anticancer efects et al. 1994). Terpenes are a large group of structurally on mammalian cells. Two examples of fusicoccanes with diverse hydrocarbons derived from the 5-carbon con- medicinal value are the diterpenoid glucosides fusicoccin taining building blocks isopentenyl pyrophosphate A and brassicicene C. Both are synthesized from FCdi- ene and were frst isolated from phytopathogenic fungi (Banerji et al. 1978; Minami et al. 2009). Fusicoccin A was found in Fusicoccum amygdali and brassicicene C was *Correspondence: rolf.wichmann@tu‑dortmund.de isolated from Alternaria brassicicola. Both fungi express 1 Laboratory of Biochemical Engineering, Department of Biochemical the same fusicocca-2,10(14)-diene synthase with identi- and Chemical Engineering, TU Dortmund University, Emil‑Figge‑Straße 66, 44227 Dortmund, Germany cal amino acid sequence (Minami et al. 2009; Arens et al. Full list of author information is available at the end of the article 2013). Due to low natural diterpenoid titers of the fungi, © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Halka et al. AMB Expr (2018) 8:132 Page 2 of 12 the fusicocca-2,10(14)-diene synthase has been previ- glucose amount can be increased proportionally by using ously expressed in several heterologous microbial hosts, more than one glucose FeedBead ® (Jeude et al. 2006). including Escherichia coli, Aspergillus nidulans and Sac- Te glucose release kinetic of one disc was measured in charomyces cerevisiae. S. cerevisiae was found to be the water (t is the given time in hours). most promising host for FCdiene production (Arens et al. mg c = 1.34 · t0.77 2013). releasedglucoseamount disc Batch fermentations in a 2 L stirred tank reactor have previously shown FCdiene concentrations of approxi- mately 10 mg/L (Arens et al. 2014). Tis has been opti- For all experiments, the synthetic dropout medium mized in our group up to around 100 mg/L FCdiene (SD medium) from Arens et al. (2014) was modifed. All while using a 3.1 L stirred tank reactor (Halka and Wich- amino acid concentrations were doubled and 95 mg/L mann 2018). Tis work presents the investigation of MgCl2 (Merck KGaA, Darmstadt, Germany) were added. glucose feeding strategies aimed at improving FCdiene All components including glucose solutions were steri- yields in fed-batch fermentations. Te strategy is a modi- lized by fltration (PES membrane, 0.2 µm). Tis medium fed protocol based on previously reported processes for composition had a pH of ~ 4.0. amorpha-4.11-diene and artemisinin production (New- man et al. 2006; Tsuruta et al. 2009; Westfall et al. 2012). Stirred fermentations S. cerevisiae growth is known to be more robust in fed- A 3.1 L glass KLF 2000 fermenter (Bioengineering AG, batch mode because of the suppressed Crabtree efect Wald, Switzerland) was used for stirred fermentations and consequent repression of ethanol production (de with a total working volume of 1.8 L of sterile fltered Deken 1966). Presented in this work is the development SD medium. Te fermenter was equipped with two six- of a model-based glucose feeding strategy which led to bladed disk turbine impellers (outer diameter: 48 mm, a consistent glucose concentration in the fermentation agitation blade: 15 mm × 12 mm) and four bafes. Te medium which was maintained at concentrations lower temperature was held at 30 °C and the pH was meas- than 0.26 g/L throughout the fermentation. In addition, ured in-line. Airfow was regulated at 0.3 LN/min using a bi-modal cultivation strategy was investigated which a mass-fow controller (MASS-VIEW®, Bronkhorst combines fed-batch followed by batch cultivation modes. High-Tech B.V., Ruurlo, Te Netherlands). Te dissolved By starting with a high biomass concentration from the oxygen tension (DOT) was measured with a DO-sensor fed-batch phase and ethanol consumption from Crabtree (Bioengineering AG, Wald, Switzerland). Oxygen, carbon efect, this strategy promoted further increases in FCdi- dioxide and ethanol concentrations in the exhaust gas ene yield per substrate. were measured online using three gas sensors (BCP-CO2, BCP-O2 and BCP-EtOH BlueSens gas sensor GmbH, Materials and methods Herten, Germany). Preparations of precultures were performed in 1 L Te strain used in this work [S. cerevisiae CEN.PK2-1C, unbafed Erlenmeyer fasks with an operating volume available at EUROSCARF (30000A) modifed from Arens of 200 mL and an initial glucose concentration of 0.14 M et al. (2014)] is engineered to mimic the one currently (25 g/L) for 24 h. used for production of artemisinin (Donald et al. 1997), with a truncated HMG-CoA reductase (tHMGR) as well Batch fermentation as the UPC2.1 mutation for increased fux to terpenes. DOT was controlled manually in order to determine the optimal aeration parameters during a fermenta- Shake fask experiments tion to avoid unintended efects on the measurement of For shake fask experiments, 300 mL Erlenmeyer fasks exhaust gas. Initially a stirring rate of 150 rpm was used. were used with a working volume of 50 mL. Te fasks Te fermentation was inoculated using an initial CDW of were incubated in a shaking incubator at 30 °C and 0.12 g/L and 0.14 M (25 g/L) glucose. 120 rpm with shaking amplitude of 2.5 cm. All shake fask experiments lasted 96 h and were performed with four Development of a feed strategy glucose FeedBeads® (Adolf Kühner AG, Basel, Switzer- Using optimal aeration parameters determined in the land) per fask. FeedBeads® are slow release discs consist- batch fermentation described above, the airfow was set ing of polymer particles made from silicon matrix-bound to 0.3 LN/min and the stirring rate was set to 500 rpm to glucose with defned release kinetics, FeedBeads have avoid oxygen limitations. An initial glucose concentra- already been verifed for promoting efective and highly tion of 0.006 M (1 g/L) was used for the frst fed-batch reproducible release kinetics in literature. Te released fermentation to investigate the glucose consumption Halka et al. AMB Expr (2018) 8:132 Page 3 of 12 during the lag-phase. After complete glucose consump- 2018). Te initial stirring rate was set to 350 rpm. Te tion, glucose was fed in pulses manually. Indicator for a glucose feed, 2.78 M (500 g/L), was supplied using a glucose pulse was an increasing DOT signal as well as a Legato™ 210P Syringe Pump (KD Scientifc Inc., Hollis- decreasing CO2 signal in the exhaust gas. A sample was ton, Massachusetts, USA). Te fermentations were inoc- taken and the actual glucose concentration was estimated ulated using an initial CDW between 0.12 and 0.2 g/L. using a rapid glucose test (Medi-Test Glucose, Mach- Te initial CDW was adapted to the feed profle because erey–Nagel, Düren, Germany). Te volume of the con- of minimum volume fow rate restriction of the syringe centrated glucose solution 2.78 M (500 g/L) to be added pump. in order to reach a concentration of maximum 0.1 g/L in the fermentation medium was calculated and added.