Elevated Î²-carotene synthesis by the engineered Rhodobacter

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Biotechnology and Biological Transformations

Elevated #-carotene synthesis by the engineered Rhodobacter sphaeroides with enhanced CrtY expression Shan Qiang, An Ping Su, Ying Li, Zhi Chen, Ching Yuan Hu, and YongHong Meng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02597 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Journal of Agricultural and Food Chemistry

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Elevated β-carotene synthesis by the engineered Rhodobacter sphaeroides with

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enhanced CrtY expression

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Shan Qiang,†,§ An Ping Su,† Ying Li,‡ Zhi Chen,‡ Ching Yuan Hu,£ Yong Hong

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Meng*,†

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†, Shaanxi Engineering Lab for Food Green Processing and Security Control, College

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of Food Engineering and Nutritional Science, Shaanxi Normal University, 620 West

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Chang’an Avenue, Chang’an, Xi’an 710119, P. R. China.

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§, Xi’an Healthful Biotechnology Co., Ltd., HangTuo Road, Chang’an, Xi’an 710100,

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P. R. China.

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‡, State Key Laboratory of Agrobiotechnology, China Agricultural University, No.2

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Yuanmingyuan West Road, Haidian District, Beijing 100193, P. R. China.

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£, Human Nutrition, Food, and Animal Science, University of Hawai'i at Manoa, 1955

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East-West Road, AgSci. 415J, Honolulu 96822-2217, USA.

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* Corresponding author:

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Yonghong Meng, Tel.: +086 029 85310517, E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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-carotene is a precursor of vitamin A and a dietary supplement for its antioxidant

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property. Producing -carotene by microbial fermentation has attracted much

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attention to consumers' preference for the natural product. In this study, an engineered

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photosynthetic Rhodobacter sphaeroides producing -carotene was constructed by the

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following strategies: (1) Five different strengths of promoters were used to investigate

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the effect of the expression level of crtY on β-carotene content. It was found that PrrnB

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increased β-carotene content by 109%. (2) Blocking of the branched pentose

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phosphate pathway by zwf deletion, and (3) Overexpressing dxs could restore the

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transcriptional levels of crtE and crtB. Finally, the engineered RS-C3 has the highest

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β-carotene content of 14.93 mg/g DCW among all the reported photosynthetic

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bacteria, and the β-carotene content reached 3.34 mg/g DCW under light conditions.

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Our results will be available for industrial use to supply a large quantity of natural β-

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carotene.

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Keywords: photosynthetic bacteria; Rhodobacter sphaeroides; β-carotene; crtY gene;

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promoters

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Introduction -carotene is widely used as a dietary supplement because it is the primary

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precursor for vitamin A, and it is an antioxidant with anti-cancer properties.1,2 More

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than 90% of commercially available -carotene is currently obtained through

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chemical synthesis.3 With consumers' preference for products from the natural sources,

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microbial fermentation has attracted much interest in the production of -carotene

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because it is an environmentally friendly and economically advantageous method

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compared with the chemical synthesis method.4

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The natural carotenogenic microorganism Blakeslea trispora has been used to study

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industrial production of -carotene.5 However, genetic manipulation is not easy in B.

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trispora, and production of natural carotenoids has been focused on optimizing the

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fermentation conditions.6,7 With the rapid development of metabolic engineering

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techniques, expression of heterologous -carotene biosynthetic genes in other model

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strains is considered as a promising strategy for -carotene biosynthesis. Many -

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carotene producers such as Escherichia coli, Saccharomyces cerevisiae, and Yarrowia

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lipolytica have been used to produce -carotene through genetic engineering.8,9,10 The

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-carotene concentration had achieved up to 4 g/L in engineered Y. lipolytica.10 The

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strains mentioned above use a chemically defined medium or complex medium to

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produce -carotene. Rhodobacter sphaeroides is a purple photosynthetic bacterium, 3



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which has attracted attention as a potential host to produce compounds under

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photosynthetic conditions. Rb. sphaeroides has been used for industrial production of

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coenzyme Q1011 and fatty acid.12 Rb. sphaeroides has a photosynthetic gene cluster of

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the seven native crt genes involved in the carotenoid synthesis, which meet the basic

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requirements of genes for -carotene biosynthesis and thus minimize the introduction

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of heterologous genes.6 Rb. sphaeroides contains a rich membrane system which is

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helpful to aggregate the -carotene in vivo.13 Chi et al. showed that the light-

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harvesting complex 2 (LH2) embedded in intracytoplasmic membranes of the

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ΔcrtI::crtIPa ΔcrtC strain contained 91% lycopene.13 Therefore, we chose Rb.

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sphaeroides as host strain and aimed to establish a β-carotene producing strain.

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Lycopene cyclase (CrtY) is the key enzyme involved in the cyclization reaction

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from lycopene to β-carotene in bacterial metabolism. The crtY gene of Pantoea

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agglomerans has been cloned and well utilized in E. coli.8 The precise control of

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gene expression is a crucial step for metabolic engineering, in which key pathway

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enzymes can help to maximize the production of desired products.14 Promoters play

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an essential role in controlling gene expression because they mainly regulate genetic

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transcription.14,15 Xu et al. optimized the expression of the E. cloacae gene cluster by

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using different strength of promoters, including PT7, Ptac, Pc, and Pabc to improve 2,

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3-butanediol production and reduce the metabolic burden in E. coli. Then E. coli 4


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BL21/pET-RABC with Pabc as a superior promoter was successfully screened.16

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Thus, optimization of the promoter strength is a critical step for heterologous gene

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expression.16 The promoters BBa_J95025, BBa_J95026, and BBa_J95027 have been

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shown to work in Rb. sphaeroides. Promoter BBa_J95025 is constitutively derived

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from the rRNA operon structure of Rb. sphaeroides. Composite promoters

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BBa_J95026 and BBa_J95027 are hybrid promoters derived from the rRNA operon

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and the lactose inducible operon. A tac promoter based constitutive expression

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plasmid was constructed for expressing the UbiG to increase the CoQ10 production

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in Rb. sphaeroides.17 In addition, a native ribosome RNA operon promoter rrnB can

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be recognized by Rb. sphaeroides. The five promoters mentioned above have

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different strengths, which can be used in Rb. sphaeroides.

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Strategies have been used to improve the yield of carotenoid production by

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increasing the supply of essential precursors through deletion or overexpression of

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upstream pathway genes.18 In the upstream of the β-carotene biosynthetic pathway of

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Rb. sphaeroides, glucose-6-phosphate dehydrogenase (Zwf) and 1-deoxy-D-xylulose-

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5-phosphate synthase (Dxs) are the two enzymes considered to have a significant

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effect on isoprenoid flux.18,19 Previously, we have demonstrated that deletion of zwf

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combined with the integrated expression of dxs could effectively enhance the

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metabolic flux and increase the yield of the lycopene production in Rb. sphaeroides.6 5



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Therefore, we would like to explore the feasibility of improving -carotene

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production through zwf deletion and dxs integration.

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In this study, we constructed and improved -carotene production by optimizing

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the promoter of the crtY, zwf knockout, and dxs integration in Rb. sphaeroides. We

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have achieved the highest β-carotene production among all the photosynthetic bacteria

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reported in the literature. We also conducted preliminary studies of producing β-

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carotene under light conditions by the engineered bacteria. The strategies used in this

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study are useful for the production of β-carotene and other carotenoids by the

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engineered photosynthetic bacteria.

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Materials and methods

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Strains and media. Table 1 listed all strains used in this study. E. coli DH5α was

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used for gene cloning. E. coli S17-1 was used for diparental conjugation. E. coli and

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Pantoea agglomerans were cultivated in Luria-Bertani (LB) medium at 37 °C, 220

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rpm supplemented with 50 μg/mL of kanamycin when necessary. For routine

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cultivation, Rb. sphaeroides was cultivated in M22+ medium containing 25 μg/mL of

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kanamycin when necessary.13 For β-carotene production under dark conditions, Rb.

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sphaeroides was cultivated in a rich medium and incubated at 34 °C, 150 rpm in 250

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mL Erlenmeyer flasks containing 50% fermentation medium and 2% inoculum with

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25 μg/mL of kanamycin when necessary.6 For β-carotene production under light

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conditions, the method was performed by Li et al. (Supporting Information)20

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Construction of plasmids. All plasmids and primers used in this study are listed in

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Table 1 and Table S1, respectively. The crtY gene derived from the P. agglomerans

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genome was amplified with primers crtYpa-F/R. The crtY gene was inserted at

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BamHI/HindIII site of plasmid pIND421 under control of the native promoter PA1/04/03

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to construct expression plasmid pIND4-crtYpa. The episomal plasmid pIND4 carried a

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kanamycin resistance gene. The codon-optimized crtY gene (opt crtY) containing

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BBa_J95025, BBa_J95026, BBa_J95027, tac (from pGEX-4T-1 plasmid) and rrnB

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promoters were synthesized by GenScript (Nanjing, China). The opt crtY sequence

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was shown in Table S2. BBa_J95025, BBa_J95026, and BBa_J95027 promoters’

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sequences were obtained from the Registry of Standard Biological Parts

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(http://parts.igem.org/Main_Page). The synthetic five opt crtY genes were inserted

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into plasmid pIND4 at EcoRI/HindIII site, and simultaneously the gene lacIq was also

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removed.17 A fusion StrepII tag for Western blot was added to the 3′ end of opt crtY

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gene.22 The synthetic opt crtY gene was PCR-amplified with primers opt crtY-tag-F/R

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and inserted into plasmid pIND4-opt crtY at NdeI/KpnI site to form plasmid pIND4-

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opt crtY-StrepII. The gene integration plasmid pK18-△crtF::opt crtY (Supporting

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Information) was constructed according to Su et al.6 7



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Diparental conjugation. Diparental conjugation was performed as described23 using

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E. coli S-17 for introducing expression plasmids pIND4 containing the crtY gene

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constructs into Rb. sphaeroides RS-L1, which is a lycopene-producing strain6. The

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plasmids pK18-△crtF::opt crtY, pK18-△zwf, and pK18-△zwf::dxs were transferred by

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conjugation from E. coli S-17 to the Rb. sphaeroides strains, as described by Su et al.6

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Genomic integrations and gene deletions were verified by diagnostic PCR and DNA

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sequencing. The detailed method can be found in Supporting Information.

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Western blot assay. The Western blot assay was performed by Matthäus et al.22 with

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minor modifications. Briefly, 10 μg total protein samples were separated by 10%

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SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was washed

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with TBST (150 mM NaCl, 10 mM Tris/HCl [pH 7.5], 0.05% [v/v] Tween 20),

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blocked with skim milk powder (5% [w/v] in TBST), and then incubated at 4 °C for

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12 h with rabbit anti-Strep-tag II antibody (1:1000 in primary antibody dilution buffer;

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Abcam; Cambridge, UK), followed by a second incubation for 1 h in an goat anti-

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rabbit (H+L) HRP (1:10000 in HRP-conjugated antibody dilution buffer; Abbkine;

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California, USA). Then, immunoreactivity was determined by the ECL method.

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Quantitative PCR (qPCR) analysis of the related genes in the β-carotene

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synthesis pathway and measurement of NADPH. Transcriptional levels of the

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related genes in the β-carotene synthesis pathway were determined by qPCR. Total 8


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RNA was extracted using the method described by Su et al.6 The qPCR was carried

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out using the SYBR tip green qPCR super mix kit (Transgen; Beijing, China). The

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rpoZ gene was used as the internal standard.24 The relative gene expression analysis

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was performed according to Su et al.6 The NADPH was measured by a

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NADP+/NADPH assay kit with WST-8 (Beyotime; Nanjing, China) and the protein

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concentration was measured by an enhanced BCA protein assay kit (Beyotime;

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Nanjing, China) in wild-type, RS-C1, RS-C2, and RS-C3 strains.

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Extraction and measurement of carotenoids. The β-carotene was extracted as

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described previously.8 1 mL of cells was harvested by centrifugation at 10,625 × g for

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2 min after 96 h of fermentation in the engineered strains, and the β-carotene content

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was measured every 24 h in the WT and RS-C3. Samples were suspended in acetone

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(1 mL) and incubated at 55 °C for 15 min in the dark with an intermittent vortex. The

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samples were then centrifuged at 10,625 × g for 2 min. The acetone supernatants

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containing β-carotene were analyzed using high-performance liquid chromatography

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(HPLC, Agilent Technologies 1260 Infinity Series system; California, USA) equipped

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with a UV detector at 450 nm and a C18 column (4.6 mm×250 mm). The mobile

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phase included acetonitrile: methanol: isopropanol (50:30:20 v/v/v) with a flow rate

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of 1 mL/min at 30 oC.7 A standard curve of different concentrations of β-carotene

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(Sigma-Aldrich) was used. The spheroidenone content was calculated by the 9



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extinction coefficient, which was 122 mM−1 cm−1 at 482 nm in 7:2

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acetone:methanol.13 The dry cell weight (DCW) of Rb. sphaeroides was measured as

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described in our previous study.6 The glucose concentration was measured by an

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SBA-40C bio-analyzer (Shandong Academy of Sciences; Jinan, China). The

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determination of malic acid was measured by Scherer et al. (Supporting

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Information)25

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Identification of β-carotene. The purification of the β-carotene sample was

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conducted according to Su et al.6 The β-carotene was identified using FTIR and

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NMR.26 The FTIR experiment was carried out using an FTIR Spectrometer TENSOR

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27 (Bruker, Germany) in the spectral range 4000−400 cm−1. 1H NMR (500 MHz) and

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13C

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spectrometer (Bruker, Germany) in CDCl3.

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Statistical analysis. Statistical analyses were conducted using SPSS 18.0 (Chicago,

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IL, USA). All experiments were repeated three times. Data in Figures 2, 4, 5, 6 and 7

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are shown as Mean ± SD. Data in Figure 2 and 4 were analyzed using one-way

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ANOVA, and LSD was used to separate the means.

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Results

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Construction of a β-carotene biosynthesis pathway in Rb. sphaeroides. The β-

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carotene biosynthesis pathway and the optimization strategies of the promoter

NMR (125 MHz) experiments were carried out using an ASCEND 600

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strength of Rb. sphaeroides are shown in Figure 1. The basal lycopene-producing

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strain RS-L1 was generated by replacing CrtI3 with CrtI4 from Rhodospirillum

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rubrum in our previous study.6 To achieve the conversion of lycopene to β-carotene in

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Rb. sphaeroides, the crtY gene from P. agglomerans was employed. Therefore, the

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episomal expression plasmid pIND4-crtY was constructed and firstly transformed into

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Rb. sphaeroides RS-L16, resulting in stain RS-L1 pC0. However, HPLC analysis

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indicated that the intermediate product lycopene in engineered strain RS-L1 pC0

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accumulated (Figure 2A). Also, the strain RS-L1 pC0 produced 4.86 mg/g DCW β-

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carotene (Figure 2B). This may be because of the relatively low expression of the

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enzyme CrtY, which was not sufficient to convert all precursor lycopene to β-carotene

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in Rb. sphaeroides RS-L1 strain6. The crtY gene was first codon-optimized for better

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expression in Rb. sphaeroides to solve this problem.

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To further optimize downstream metabolic pathway from lycopene to β-carotene,

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five different strengths of promoters were selected to identify the optimal expression

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level of the gene opt crtY. To express the heterologous gene opt crtY, five plasmids

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(pIND4-PJ95025-opt crtY, pIND4-PJ95026-opt crtY, pIND4-PJ95027-opt crtY, pIND4-Ptac-

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opt crtY, and pIND4-PrrnB-opt crtY harboring promoters—J95025, J95026, J95027,

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tac, and rrnB promoter, respectively) were constructed and constitutively expressed.

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All these plasmids were based on the plasmid pIND4 in which the lacIq cassette was 11



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eliminated.17 All these plasmids were transformed into RS-L1, resulting in strains RS-

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L1 pC1, RS-L1 pC2, RS-L1 pC3, RS-L1 pC4, and RS-L1 pC5, respectively. After 96

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h of fermentation under dark conditions, β-carotene content was analyzed using

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HPLC and compared. As shown in figure 2B, the RS-L1 strains harboring pC1, pC2,

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pC3, pC4, and pC5 produced 9.65 mg/g DCW, 7.26 mg/g DCW, 8.63 mg/g DCW,

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9.04 mg/g DCW, and 10.17 mg/g DCW of β-carotene, respectively (P

Elevated Î²-carotene synthesis by the engineered Rhodobacter

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