Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling.

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L-1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L-1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L-1 (99.3%) and 75.1 ± 2.5 g·L-1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions

Rewriting the metabolic blueprint: advances in pathway diversification in microorganisms

10.3389/fmicb.2018.00155Frontiers in Microbiology9FEB155completedcomplete

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered <i>Escherichia coli</i>: Process Optimization and Kinetics Modeling - Fig 3

<p>Time profiles of PPA production using growing and resting cell biotransformation in the flask (a) and 3-L bioreactor (b).</p

Cellular localization and substrate specificity.

<p>(A) Activity of l-AAD in the different cellular location; (B) Substrate specificity of l-AAD after heterologous expression in the <i>E. coli</i>. The highest activity of the l-AAD biocatalyst with l-methionine was defined as 100%.</p

Comparison of <i>V</i>_<i>max</i> and <i>K</i>_<i>m</i> of the resting cell biotransformation with different PPA concentrations.

<p>Comparison of <i>V</i>_<i>max</i> and <i>K</i>_<i>m</i> of the resting cell biotransformation with different PPA concentrations.</p

One-Step Biosynthesis of α-Keto-γ-Methylthiobutyric Acid from L-Methionine by an <i>Escherichia coli</i> Whole-Cell Biocatalyst Expressing an Engineered L-Amino Acid Deaminase from <i>Proteus vulgaris</i>

<div><p>α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an <i>Escherichia coli</i> whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from <i>Proteus vulgaris</i>. We first overexpressed the <i>P. vulgaris</i>l-AAD in <i>E. coli</i> BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl₂, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct <i>P. vulgaris</i>l-AAD mutant libraries. Among approximately 10⁴ mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca²⁺ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.</p></div

Kinetic analysis of cell deactivation, substrate and product inhibition of the resting cell biotransformation in the 3-L bioreactor.

<p>a: time profiles of dissolved oxygen with different l-phenylalanine concentrations. b: time profiles of cell deactivation at different l-phenylalanine concentrations. c: calculation of the deactivation constant. d: effect of biocatalyst concentration on the initial rate. e: effect of substrate concentration on the initial reaction rate. f: Lineweaver-Burk plotting with different PPA addition.</p