Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction

Mr, Gerigk; Maass, Danielle; A, Kreutzer; Sprenger, Georg A.; Bongaerts, Johannes; Wubbolts, Marcel; Takors, Ralf

doi:10.1007/s00449-002-0280-2

Cited by 44 publications

(5 citation statements)

References 65 publications

(65 reference statements)

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“…When necessary, the following concentrations of antibiotics were added to the medium to maintain plasmids: 100 μg/mL ampicillin (Amp) and 40 μg/mL kanamycin (Kan). Flask cultivation for the production of l -phenylalanine was performed using modified fermentation medium previously [ 51 – 53 ]. The composition of the fermentation medium is: 20 g/L glucose, 3 g/L KH 2 PO 4 , 5 g/L (NH 4 ) 2 SO 4 , 1 g/L NaCl, 1.5 g/L sodium citrate, 0.015 g/L CaCl 2 ∙2H 2 O, 3 g/L MgSO 4 ∙7H 2 O, 0.01125 g/L FeSO 4 ∙7H 2 O, 0.075 g/L Thiamine-HCl, 0.3 g/L l -tyrosine (L-Tyr), 0.03 g/L l -tryptophan (L-Trp), 3 g/L yeast extract, and 1.5 mL/L trace element solution (TES) at pH 6.8.…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

et al. 2016

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BackgroundPlant parasitic nematodes are harmful to agricultural crops and plants, and may cause severe yield losses. Cinnamaldehyde, a volatile, yellow liquid commonly used as a flavoring or food additive, is increasingly becoming a popular natural nematicide because of its high nematicidal activity and, there is a high demand for the development of a biological platform to produce cinnamaldehyde.ResultsWe engineered Escherichia coli as an eco-friendly biological platform for the production of cinnamaldehyde. In E. coli, cinnamaldehyde can be synthesized from intracellular l-phenylalanine, which requires the activities of three enzymes: phenylalanine-ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and cinnamoyl-CoA reductase (CCR). For the efficient production of cinnamaldehyde in E. coli, we first examined the activities of enzymes from different sources and a gene expression system for the selected enzymes was constructed. Next, the metabolic pathway for l-phenylalanine biosynthesis was engineered to increase the intracellular pool of l-phenylalanine, which is a main precursor of cinnamaldehyde. Finally, we tried to produce cinnamaldehyde with the engineered E. coli. According to this result, cinnamaldehyde production as high as 75 mg/L could be achieved, which was about 35-fold higher compared with that in the parental E. coli W3110 harboring a plasmid for cinnamaldehyde biosynthesis. We also confirmed that cinnamaldehyde produced by our engineered E. coli had a nematicidal activity similar to the activity of commercial cinnamaldehyde by nematicidal assays against Bursaphelenchus xylophilus.ConclusionAs a potential natural pesticide, cinnamaldehyde was successfully produced in E. coli by construction of the biosynthesis pathway and, its production titer was also significantly increased by engineering the metabolic pathway of l-phenylalanine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0415-9) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

et al. 2016

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“…The production of L-phenylalanine by various recombinant E. coli strains using glucose or sucrose as carbon sources has been discussed extensively in the literature [2,8,9,14,19,26,31,32], but reports on the use of glycerol for this fermentation are nonexistent.…”

Section: Introductionmentioning

confidence: 99%

Production of l-phenylalanine from glycerol by a recombinant Escherichia coli

Khamduang

Packdibamrung

Chutmanop

et al. 2009

J Ind Microbiol Biotechnol

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The production of L-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing L-phenylalanine using the genetically modiWed bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a deWned medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300-500 rpm corresponding to 0.97-1.62 m s ¡1 impeller tip speed) and aeration rates (2-8 L min ¡1 , or 1-4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed »1.62 m s ¡1 ) reduced the biomass and L-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of L-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s ¡1 impeller tip speed, 4 vvm aeration rate), the yield of L-phenylalanine on glycerol was 0.58 g g ¡1 , or more than twice the best yield attainable on sucrose (0.25 g g ¡1 ). In the best case, the peak concentration of L-phenylalanine was 5.6 g L ¡1 , or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of L-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose-and glucosebased fermentations.

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“…In development of industrial process, E. coli gives the advantages of high growth and production rates combined with high yields by using simple media which are suitable for product recovery efficiently (Gerigk et al, 2002). Hence, using recombinant E. coli strains is a great deal on the L-Phe process development and many research works have been patented (Kim et al, 1994, Lim et al, 1995, Sprenger et al, 1998.…”

Section: L-phenylalanine Productionmentioning

confidence: 99%

“…This enzyme is also sensitive to feedback inhibition mediated by allosteric binding of L-Phe (Pittard, 1996, Zhang et al, 1998. Gerigk and coworkers used recombinant E. coli carrying feedback-resistant pheA (pheA fbr ) and feedback-resistant aroF (aroF fbr ) to synthesize L-Phe in 20 L bioreactor and achieved more than 30 g/L of L-Phe (Gerigk et al, 2002). It also has been reported that the construction of genetically modified E. coli strain with pheA fbr and aroF fbr , with L-Phe titer of 50 g/L was achieved after 36 h (Backman et al, 1990).…”

Section: Metabolic Engineering For Biosynthesis Of L-phenylalanine In...mentioning

confidence: 99%

“…Liu Ratchaneeladdajit, (2014), the production of L-Phe by recombinant E. coli was significantly improved by expression of aroG fbr in the pBLPTG. In addition, the titer and yield of L-Phe can be improved by optimizing the fermentation parameters (Gerigk et al, 2002, Zhou et al, 2011, or performing in situ product recovery (Ruffer et al, 2004). showed the highest DAHP synthase activity in the present of 5 mM L-Phe (Yenyuvadee, 2016).…”

Section: Plasmid Extraction and Restriction Enzyme Digestion Analysismentioning

confidence: 99%

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Effect of feedback-resistant aroG overexpression on L-phenylalanine production in Escherichia coli

Ulfah

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L-Phenylalanine (L-Phe) is one of the most important amino acids in food and pharmaceutical industries. It is widely used as a nutritional supplement and a precursor for the synthesis of food additives. The market of L-Phe has been stimulated by increasing demand for the low-calorie sweetener, aspartame.� In�Escherichia coli,�the key enzyme which catalyzes the first committed step of aromatic amino acid biosynthesis pathway is 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHP-synthase).�The enzyme has 3 isoforms,�AroG, AroF and�AroH, which are feedback inhibited by L-Phe, L-Tyr and L-Trp, respectively. AroG, encoded by�aroG, is the major isoform contributed about 80% of the total DAHP activity. To investigate the feedback inhibition site of AroG, Leu175 was replaced by Asp (L175D) and Gln151 was replaced by Ala (Q151A), Leu (Q151L) and Asn (Q151N). In this study, each feedback resistant�aroG�was cloned with other pivotal genes in L-Phe biosynthesis pathway (aroB, aroL, phedh�and�tktA) into pRSFDuet-1 vector. The recombinant plasmid (pBLPTG*) were then co-expressed with pBAD33 vector containing a glycerol uptake gene (glpF) and an aromatic amino acid exporter gene (yddG) (pYF) into�E.�coli�BL21(DE3). The highest production of L-Phe at 1.8 g/L was obtained when both pBLPTG*Q151L & pYF and pBLPTG*Q151N & pYF clones were cultured in glycerol medium for 6 days. These L-Phe yields were 7.7 fold higher than obtained from the control with�aroG�wild-type (pBLPTG & pYF), while L-Phe yield from pBLPTG*L175D & pYF and pBLPTG*Q151A & pYF were 2.7 and 2.5 fold, respectively. The results revealed that substitution of Leu and Asn at Gln151 could well reduce�the feedback inhibition. After that the recombinant clone of pBLPTG*Q151L & pYF was selected for optimization of medium components using response surface methodology (RSM). The maximum L-Phe production at 2.03 g/L was obtained when the pBLPTG*Q151L & pYF was cultured in minimum medium containing�60 g/L glycerol and 42.4 g/L (NH4)2SO4. after induction with 0.02% arabinose at 37 ?C for 6 days.

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Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction

Cited by 44 publications

References 65 publications

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

Production of l-phenylalanine from glycerol by a recombinant Escherichia coli

Effect of feedback-resistant aroG overexpression on L-phenylalanine production in Escherichia coli

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