Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 μM cinnamic acid per unit of optical density at 600 nm [OD(600)]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 μM p-coumaric acid OD(600) unit(−1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases

Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport

Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively

Increasing the NADPH supply for whole-cell biotransformation and development of a novel biosensor

In the first part of this work, the pentose phosphate pathway (PPP) was investigated as a source of NADPH in reductive whole-cell biotransformation using $\textit{Escherichia coli}$ and $\textit{Corynebacterium glutamicum}$ as hosts and glucose as reductant. The reduction of methyl acetoacetate to the chiral (R)-methyl hydroxybutyrate (MHB) served as a model reaction for NADPH-dependent reactions and was catalyzed by an alcohol dehydrogenase (ADH) from $\textit{Lactobacillus brevis}$. Partial cyclization of the PPP in $\textit{E. coli}$ and $\textit{C. glutamicum}$ was achieved by deletion of the phosphofructokinase gene $\textit{pfkA}$, which prevents fructose 6-phosphate catabolism in the glycolytic pathway. The $\textit{pfkA}$-deficient mutants carrying the $\textit{L. brevis}$ ADH showed a doubled MHB-per-glucose ratio compared to the parent strains. In $\textit{E. coli}$ the partial PPP cyclization in the Δ$\textit{pfkA}$ mutant was proven by $^{13}$C-flux analysis, which showed a negative net flux through the phosphoglucose isomerase reaction. Furthermore, the flux through pyruvate kinase was found to be absent in the Δ$\textit{pfkA}$ mutant, indicating that a low phosphoenolpyruvate (PEP) concentration limited glucose uptake via the phosphotransferase system (PTS). PTS-independent glucose uptake and phosphorylation via the glucose facilitator and glucose kinase from $\textit{Zymomonas mobilis}$ enhanced the specific MHB productivity by 21% in the $\textit{E. coli ΔpfkA}$ mutant. Deletion of glyceraldehyde 3-phosphate dehydrogenase ($\textit{gapA}$) theoretically results in a completely cyclized PPP and a ratio of 12 mol NADPH per mol glucose 6-phosphate. A $\textit{C. glutamicum ΔgapA}$ mutant showed a ratio of 7.9 mol MHB per mol glucose, which is the highest one reported so far. Formation of the by-product glycerol presumably was responsible for not achieving a higher ratio. In the second part of this work, a biosensor was developed which is capable of detecting a lowered intracellular NADPH/NADP+ ratio and trigger the synthesis of an autofluorescent protein. DNA microarray analysis of $\textit{E. coli}$ during biotransformation showed an upregulation of $\textit{soxS}$ transcription after MAA addition, suggesting that the SoxR regulator known to upregulate soxS expression is activated by a lowered NADPH/NADP$^{+}$ ratio. Subsequently, the $\textit{soxS}$ promoter was fused on a plasmid with the gene encoding yellow fluorescent protein (eYFP). $\textit{E. coli}$ transformed with this plasmid showed fluorescence when MAA was added to the culture. The final fluorescence [...