Production of trans-cinnamic acid by whole-cell bioconversion from l-phenylalanine in engineered Corynebacterium glutamicum

Jaewoo Son,In Hyeok Choi,Jun Hong Jang,Ki Jun Jeong,Eun Jung Jeon,Hyun Bae Bang,Chang Gyu Lim

doi:10.1186/s12934-021-01631-1

Jaewoo Son, In Hyeok Choi + Show 5 more

Open Access

https://doi.org/10.1186/s12934-021-01631-1

Copy DOI

Abstract

Backgroundtrans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of l-phenylalanine (l-Phe) into t-CA and developed a repeated bioconversion process.ResultsAn expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of l-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of l-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation.ConclusionsWe developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

Highlights

Expression of the Phenylalanine ammonia lyase from Streptomyces maritimus (SmPAL) gene in C. glutamicum for t‐CA production Previously, we examined the efficiencies of a few Phenylalanine ammonia lyase (PAL) enzymes for the biosynthesis of trans-cinnamic acid (t-CA) in E. coli, and SmPAL showed the highest production of t-CA [36]
Unlike other PALs, SmPAL is known to have relatively low TAL activity leading to p-coumaric acid instead of t-CA [37, 38]
We found that the t-CA production yield decreased gradually (Fig. 5b), and this may be ascribed to the loss of activity of SmPAL

Summary

Methods

E. coli XL1-Blue was used for gene cloning and plasmid maintenance, and C. glutamicum ATCC 13,032 was used as the main host for bioconversion. E. coli XL1-Blue was cultivated in Luria-Bertani medium (BD, Franklin Lakes, NJ, USA) at 37 °C and 200 rpm. C. glutamicum was cultivated in CGXII medium (3 g/L K2HPO4, 1 g/L K H2PO4, 2 g/L urea, 10 g/L (NH4)2SO4, 2 g/L MgSO4, 200 μg/L biotin, 5 mg/L thiamine, 10 mg/L calcium pantothenate, 10 mg/L F eSO4, 1 mg/L M nSO4, 1 mg/L ZnSO4, 200 μg/L C uSO4, 10 mg/L C aCl2 and Plasmid construction For gene expression in C. glutamicum, pHCMS, the highcopy number plasmid derivative of pCES208, was used as the backbone plasmid [39]. To express SmPAL, the native SmPAL gene was amplified from pHB-104 [36] by PCR using primers F-T7R-SmPAL and R-SmPAL (Additional file 4: Table S1). The PCR product was digested with restriction enzymes XbaI and NotI and cloned into pHCMS, yielding pHCMS-SmPAL, in which the SmPAL gene was constitutively expressed under the strong synthetic promoter PH36 [40]

Results

Discussion

Conclusion