Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli.

Minjae Han,Pyung Cheon Lee

doi:10.3390/microorganisms9071520

Abstract

Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15′-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5’-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid–producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli.

Highlights

Retinoids are essential components of visual function, cell differentiation, and other cellular signaling pathways [1,2]
Retinoic acid is biosynthesized from β-carotene in two reaction steps (Figure 1). β-carotene is symmetrically cleaved by β-carotene 15,150 -oxygenase (Blh), generating retinaldehyde, which is oxidized to retinoic acid by retinal dehydrogenase (Raldh)
When the blhSR and raldhHS genes were induced through the 6×His tagging system [pET21α(+) plasmid] in E. coli BL21 (DE3), one band corresponding to each gene was detected during immunoblotting (Figure 2A)

Summary

Introduction

Retinoids (or vitamin A and its analogs) are essential components of visual function, cell differentiation, and other cellular signaling pathways [1,2]. Retinoids are lipophilic compounds with diverse structures, based on their end groups. They are composed of three structural moieties: a β-ionone ring, an isoprenoid backbone, and a functional group such as an alcohol (retinol), an aldehyde (retinal), a carboxylic acid (retinoic acid), or an ester group (retinyl esters) [3]. Retinoic acid is one of the most important ingredients in cosmetic skincare products because it can protect against UV-radiation-induced skin damage in fibroblasts and other skin cells [5]. The retinoic acid precursor β-carotene is synthesized by four biosynthetic pathway enzymes [7], known as geranylgeranyl diphosphate synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene cyclase (CrtY) (Figure 1)

Methods

Results

Conclusion