Capsanthin Production in Escherichia coli by Overexpression of Capsanthin/Capsorubin Synthase from Capsicum annuum

Abstract

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene β-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

“…Besides iris calli, to date, capsanthin production by CCS expression was demonstrated in transgenic plants of Nicotiana benthamiana [84], Solanum lycopersicum [54] and Oryza sativa [85]. Recently the production of this pigment was achieved also in Escherichia coli strains [86]. Contrary to our findings, in leaves of Nicotiana benthamiana plants transfected with a viral RNA vector that harbored pepper ccs gene, capsanthin is accumulated also in leaves which gave orange color to the whole plant [52].…”

Promiscuous activity of β‐carotene hydroxylase CrtZ on epoxycarotenoids leads to the formation of rare carotenoids with 6‐hydroxy‐3‐keto‐ε‐ends

“…CrtZ, a b-end 2-hydroxylase, from three different bacteria results in different profile of carotenoids Escherichia coli can be engineered to biosynthesize violaxanthin by expressing bacterial zeaxanthin pathway genes and a plant-derived zeaxanthin epoxidase (ZEP) gene [11][12][13][14]. In our previous study [11] in which we used P. ananatis zeaxanthin pathway genes (crtEBIYZ) and Capsicum annuum ZEP gene (CaZEP) for violaxanthin production, we noticed that using a crtZ gene from different organisms resulted in a different byproduct profile (data not shown). To confirm and to further elucidate this serendipitous discovery, we performed the following experiment.…”

“…pACT-P J23107 -Zea-crtZ Pag-IP was constructed by amplifying crtZ Pag gene by primers oMF168/ oMF170 set and oMFseq56/oMF169 set (Table S3) using pACT-P J23107 -Zea-crtZ Pag as a template, then assembling these two fragments with NotI/KpnI-digested fragment of pACT-P J23107 -Zea-crtZ Bre . pUClac-CaZEP is derived from a previous study [11]. pUClac-P tac -crtZ B , pUClac-P tac -crtZ Pan , pUClac-P tac -crtZ Pag , and pUClac-P tac -crtZ Pag-IP are constructed by inserting the codon-optimized crtZ sequences (amplified from pACT-Zea plasmids) into the EcoRI/XhoI site of pUClac vector [11] using homology cloning.…”

“…pUClac-P tac -crtZ B , pUClac-P tac -crtZ Pan , pUClac-P tac -crtZ Pag , and pUClac-P tac -crtZ Pag-IP are constructed by inserting the codon-optimized crtZ sequences (amplified from pACT-Zea plasmids) into the EcoRI/XhoI site of pUClac vector [11] using homology cloning. pUClac-P J23115/lac -crtZ plasmids were constructed by first preparing pUClac-P J23115/lac -sfgfp vector by replacing the P tac promoter sequence of pUClac vector [11] by amplifying the whole plasmid sequence using primers oMF198 and oMF199, then ligating them using type IIS restriction enzyme assembly. Then the crtZ sequences were amplified from pACT-Zea-plasmids and inserted them into EcoRI/ XhoI site of pUClac-P J23115/lac -sfgfp.…”

Colorful Treasure From Agro-Industrial Wastes: A Sustainable Chassis for Microbial Pigment Production

“…The transformed strains, i.e., Paracoccus aminophilus CRT1 and Paracoccus kondratievae CRT2 could effectively grow on industrial effluents, i.e., flue gas desulfurization (FGD) wastewater supplemented with molasses and produced 127.09 and 58.80 ng/mL carotenoids, respectively. Using an analogous approach, Furubayashi et al (2021) reported for the first time, the microbial production of capsanthin, a characteristic carotenoid found generally in Capsicum annuum . A heterologous capsanthin bio-synthetic pathway was engineered in Escherichia coli by expressing eight genes which included five zeaxanthin biosynthesis genes from a soil bacterium ( Pantoea ananatis ), zeaxanthin epoxidase (ZEP) and capsanthin/capsorubin synthase (CCS) from C. annuum and isopentenyl diphosphate isomerase (IDI) from green alga ( Haematococcus pluvialis ).…”