Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli

Abstract

L-tyrosine is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. Although several attempts have been made to improve L-tyrosine production, translation-level expression control and carbon flux rebalancing around phosphoenolpyruvate (PEP) node still remain to be achieved for optimizing the pathway. Here, we demonstrate pathway optimization by altering gene expression levels for L-tyrosine production in Escherichia coli. To optimize the L-tyrosine biosynthetic pathway, a synthetic constitutive promoter and a synthetic 5'-untranslated region (5'-UTR) were introduced for each gene of interest to allow for control at both transcription and translation levels. Carbon flux rebalancing was achieved by controlling the expression level of PEP synthetase using UTR Designer. The L-tyrosine productivity of the engineered E. coli strain was increased through pathway optimization resulting in 3.0 g/L of L-tyrosine titer, 0.0354 g L-tyrosine/h/g DCW of productivity, and 0.102 g L-tyrosine/g glucose yield. Thus, this work demonstrates that pathway optimization by 5'-UTR redesign is an effective strategy for the development of efficient L-tyrosine-producing bacteria.

Figure 1. The L-tyrosine biosynthetic pathway engineering strategy.

(a) Each gene was under the control of synthetic expression design that substitutes native promoter and 5′-UTR with synthetic constitutive promoter and designed 5′-UTR specific to target gene. (b) Dashed lines indicate feedback regulation, ‘X’ denotes deletion of the tyrR gene, and thick red arrows represent overexpression of genes in the L-tyrosine synthetic pathway. Abbreviations: Glc, glucose; PEP, phosphoenolpyruvate; Pyr, pyruvate; AcCoA, acetyl-CoA; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHIK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 3-hydroxyphenylpyruvate; L-tyr, L-tyrosine; KPP, keto-phenylpyruvate; L-Phe, L-phenylalanine. (c) Comparison of L-tyrosine productivity of wild-type (W3110) and rationally engineered (SCK1) E. coli strain. The specific productivity of L-tyrosine was increased to 0.0014 g/h/g DCW in the SCK1 strain while the wild type strain did not produce L-tyrosine. The y-axis represents specific productivity of L-tyrosine (g/h/g DCW) in each strain. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.

Figure 2. Carbon flux redistribution at the PEP node by fine-tuning of ppsA expression.

(a) Pathway optimization for fine-tuning the expression levels of the ppsA gene using UTR Designer. J23100 indicates a strong constitutive promoter from the Registry of Standard Biological Parts (BBa_J23100; http://partsregistry.org). (b) Comparisons of predicted expression levels from UTR Designer and specific enzyme activities of ppsA variants. (c) The specific L-tyrosine productivity of each ppsA variant after 24 h cultivation in M9 minimal medium. Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures.

Figure 3. Fermentation profiles of E. coli strains cultivated in complex medium.

Data for the (a) wild-type (W3110; open symbols) and (b) SCK5 (closed symbols) E. coli strains are shown. pH adjustments were made at 6-h intervals. The left y-offset and right y-axis represent glucose and L-tyrosine concentrations (g/L), respectively. The left y-axis represents OD₆₀₀ and the x-axis represents culture time (h). Each point and error bar indicates means and standard deviations between measurements from biological triplicate cultures. Symbols: circle, OD₆₀₀; triangle, glucose; rectangle, L-tyrosine.