In this issue, we recommend an article by Professor Zhou Jingwen, Deputy Director of the Future Food Science Center at Jiangnan University, and his team published in Synthetic and Systems Biotechnology Synergetic engineering of Escherichia coli for efficient production of L-tyrosine。 This study utilized a collaborative engineering strategy to target the shikimic acid pathway and fine tune metabolic processes, and utilized laboratory adaptive evolution (MMC) to improve the strain's tolerance to acidic conditions. Ultimately, an optimized strain was obtained, which can produce 92.5 g/L L-tyrosine within 62 hours in a 5-liter fermentation tank, which is of great significance for the industrial production of L-tyrosine.

L-tyrosine has a wide range of applications in the food, feed, chemical, and pharmaceutical industries. It is not only a precursor to various natural products such as caffeic acid, flavonoids, and curcumin, but also involved in the synthesis of the drug L-DOPA for the treatment of Parkinson's disease. Although L-tyrosine has important industrial value, traditional chemical synthesis or enzymatic methods for producing tyrosine have the disadvantages of high cost and poor stability. Microbial fermentation for tyrosine production has the advantages of low cost and simple operation, but it also has disadvantages such as low yield and unstable high-density fermentation strains.

Researchers used Escherichia coli WSH-Z06 as the starting strain to perform mixed validation of overexpression and knockout of several genes related to the accumulation of shikimic acid and aromatic amino acids. A strain combining overexpression of four genes and knockout of three genes was obtained, and the L-tyrosine production in shaking flasks reached 4.22 g/L within 48 hours (Figure 2). Then, the phosphoketolase pathway (PK) was added to its shikimic acid pathway, and it was found that the strain's OD600 in shake flasks increased by 9.8%, but the overall L-tyrosine production showed a decreasing trend. After integrating two genes involved in the interconversion of NADH and NADPH, the yield of the obtained strain increased to 6.17g/L (Figure 3a). The strain was subjected to fermentation at the 5L fermenter level, and the yield of L-tyrosine reached 50.2 g/L. At the same time, 12.4 g/L of acetic acid was detected in the fermentation broth (Figure 3b). High concentrations of acetic acid significantly inhibited bacterial growth (Figure 3b).

In order to reduce the accumulation of acetic acid, the gene encoding the acetic acid production pathway in Escherichia coli was knocked out, resulting in a 33.6% decrease in acetic acid production and a slight improvement in bacterial growth. Further enhancing its tolerance to acetic acid and increasing lysine yield, adaptive evolution was carried out using MMC (Microbial Droplet Continuous Passage Evolutionary Instrument). More than 200 droplets were screened for 50 passages in the droplet system, and the acetic acid concentration was continuously increased to obtain a strain that grew well at pH 5.1, with the best shaking bottle level yield of 7.11 g/L (Figure 4). By controlling the glucose concentration and dissolved oxygen in a 5L fermentation tank, the final L-tyrosine yield reached 92.8 g/L.

This series of comprehensive engineering strategies significantly promoted the yield of L-tyrosine in microbial synthesis, providing strong experimental support for industrial production.

Figure 1 Glucose biosynthesis pathway of L-tyrosine

Figure 2 Lysine production after gene knockout and overexpression

Figure 3: Fermentation Results of Optimized Strain in Shake Bottle and 5L Fermentation Tank

Figure 4: Modification and Acid Resistance Evolution of Escherichia coli Acetic Acid System

Article link: https://doi.org/10.1016/j.synbio.2023.10.005