Metabolic Redesign of Rhodobacter sphaeroides for Lycopene

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Biotechnology and Biological Transformations

Metabolic Redesign of Rhodobacter sphaeroides for Lycopene Production Anping Su, Shuang Chi, Ying Li, Siyuan Tan, Shan Qiang, Zhi Chen, and Yonghong Meng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00855 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Journal of Agricultural and Food Chemistry

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Metabolic Redesign of Rhodobacter sphaeroides for Lycopene Production

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Anping Sua, Shuang Chib, Ying Lib, Siyuan Tana, Shan Qiangc, Zhi Chenb,*,

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Yonghong Menga,*

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a, 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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b, 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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c, Xi’an Healthful Biotechnology Co., Ltd. HangTuo Road, Chang’an, Xi’an 710100,

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P. R. China.

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* correspondence:

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

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Zhi Chen, Tel: +010 62732715, E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Lycopene plays an important role as an anti-oxidative and anti-cancer agent, and is an

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increasingly valuable commodity in the global market. Rhodobacter sphaeroides, a

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carotenogenic and phototrophic bacterium, is an efficient and practical host for

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carotenoid production. Herein, we explored the potential of metabolically engineered

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Rb. sphaeroides as a novel platform to produce lycopene. The basal lycopene-

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producing strain was generated by introducing an exogenous crtI4 from

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Rhodospirillum rubrum to replace the native crtI3 and deleting crtC in Rb.

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sphaeroides. Furthermore, knocking out zwf blocked the competitive pentose

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phosphate pathway and improved the lycopene content by 88%. Finally, the

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methylerythritol phosphate pathway was reinforced by integration of dxs combined

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with zwf deletion, which further increased the lycopene content. The final engineered

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strain produced lycopene to 10.32 mg/g dry cell weight. This study describes a new

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lycopene producer and provides insight into a photosynthetic bacterium as a host for

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lycopene biosynthesis.

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Keywords: Rhodobacter sphaeroides; Rhodospirillum rubrum; construction of

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metabolic pathway; crtI4 gene; lycopene synthesis

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Introduction

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Carotenoids are a popular class of high value-added natural tetraterpene compounds

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and pigments 1. Among the carotenoids, lycopene is one of the most efficient

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biological carotenoid singlet oxygen quenchers and plays a critical role as an anti-

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oxidative and anti-cancer agent 2-3. Because of these distinctive biological properties,

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lycopene has been used in a variety of fields including the functional food,

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nutraceutical, pharmaceutical and cosmetic industries and has garnered increasing

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demand in the global market 4. Generally, lycopene is obtained by extraction from

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plants, chemical synthesis and microbial fermentation. With the development of

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metabolic engineering and synthetic biology, microbial fermentation has proven to be

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an economic, environmental friendly and sustainable technique for lycopene

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production 1, 5.

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Currently, microbial production of lycopene employed in industrialized

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processes uses the natural carotenogenic microorganism Blakeslea trispora 6-7.

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However, the production of lycopene from B. trispora requires the addition of cyclase

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inhibitors, which are expensive and cause food safety issues 8. Additionally, genetic

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manipulation is not easy in B. trispora, thereby hindering its metabolic engineering

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for yield improvement. Rhodobacter sphaeroides, a photosynthetic bacterium,

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provides some advantages as a new platform for lycopene production. It harbors a 3 / 29



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photosynthetic gene cluster containing the seven native crt genes (crtF, crtE, crtD,

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crtC, crtB, crtI and crtA) involved in carotenoid synthesis 9, which almost meet the

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requirements of genes for lycopene biosynthesis and minimize the use of exogenous

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genes. It has a rich membrane system that favors lycopene aggregation in membranes

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10

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as those used to synthesize high value-added natural products on a large-scale

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including coenzyme Q10 11, fatty acids 12 and 5-aminolevulinic acid 13, have paved the

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way for study of Rb. sphaeroides as a model strain for biochemical production.

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Fermentation of Rb. sphaeroides is also cost-effective as it can naturally synthesize

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carotenoids under anoxygenic and photosynthetic conditions 14.

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. Furthermore, the development of well-established genetic manipulation tools, such

In the lycopene biosynthesis pathway, phytoene dehydrogenase (CrtI) is a key

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enzyme that introduces a four-step dehydrogenation via three intermediates,

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phytofluene, ζ-carotene and neurosporene, to form lycopene 15. However, the native

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phytoene dehydrogenase in Rb. sphaeroides is a three-step dehydrogenase (CrtI3),

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which catalyzes the conversion of phytoene to neurosporene (yellow pigment). Thus,

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to construct a lycopene biosynthesis pathway in Rb. sphaeroides, a four-step

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dehydrogenase (CrtI4) must be chosen to replace the native CrtI3. Here, a CrtI4 from

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Rhodospirillum rubrum was first chosen to replace the native CrtI3 to engineer a

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platform organism capable of mass-producing lycopene. Additionally, the native 4 / 29


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neurosporene hydroxylase (CrtC) is able to intervene in the catalyze of four

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desaturations, prematurely terminating the reaction of CrtI 16. Therefore, the native

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crtC must be deleted to block the lycopene-consuming pathway and redistribute the

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metabolic flux for lycopene accumulation.

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To improve the yield of target products, it is often necessary to enhance the

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supply of essential precursors. In the upstream portion of the lycopene biosynthesis

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pathway in Rb. sphaeroides, the glucose-6-phosphate dehydrogenase (Zwf), which

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resides in the central carbon metabolic pathway, was reported to be one of the key

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enzymes and plays an important role in isoprenoid flux 17. The 1-deoxy-D-xylulose-5-

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phosphate synthase (Dxs), which resides in the methylerythritol phosphate (MEP)

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pathway, was also identified as a key rate-limiting enzyme for flux delivery 18. It has

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been demonstrated that deletion of the zwf gene and overexpression of the dxs gene

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can successfully strengthen the metabolic flux and improve the production of the

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corresponding products in E. coli 19. Here, we initially engineered the lycopene

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upstream metabolic pathway by combining a zwf knockout and dxs integration in Rb.

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sphaeroides to increase the essential precursors and eventually improve the lycopene

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content.

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In brief, we engineered and characterized the ability of Rb. sphaeroides to

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produce lycopene. The lycopene content reached 10.32 mg per gram of dry cell 5 / 29



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weight (DCW), which is the highest reported content of lycopene in photosynthetic

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microorganisms. This study describes a novel lycopene producer and provides insight

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into photosynthetic bacteria as a cell factory for microbial production of lycopene and

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other carotenoids.

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Materials and methods

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Strains and culture conditions

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All strains used in this study are listed in Table 1. E. coli DH5α 20 was used for

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routine cloning procedures and E. coli S17-1 21was used for di-parental conjugation.

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Both were grown in Luria-Bertani (LB) medium at 37 °C, 220 rpm with 50 µg/mL of

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kanamycin when necessary. R. rubrum was cultivated in SMN medium 22. For routine

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cultivation, the wild type Rb. sphaeroides and subsequent recombinant strains were

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grown in M22+ medium 10.

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Construction recombinant plasmids

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The plasmids used in this study are shown in Table 1. For gene deletion, the upstream

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and downstream regions of the crtC and zwf coding regions were amplified with

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primers as described in Table S1, and then the upstream and downstream sequences

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were fused by overlapping PCR. The resulting fragments were inserted into

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pK18mobsacB to form the plasmids pK18-△crtC and pK18-△zwf. For gene

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integration, the upstream and downstream regions of the corresponding insertion sites 6 / 29


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were amplified using primers as described in Table S1, and then the upstream and

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downstream sequences were assembled with the crtI4 or dxs gene, respectively. The

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fragments were ligated into pK18mobsacB to form the plasmids pK18-△crtI3:: crtI4

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and pK18-△zwf:: dxs. All the plasmids were verified by sequencing and transferred

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into E. coli S17-1 for conjugation mating.

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Di-parental conjugation and screening of recombinant strains

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Di-parental conjugation was carried out as reported by Chi et al. 10 with some

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modifications. The homologous single exchange colonies were selected in M22+

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containing 25 µg/mL kanamycin and 2 µg/mL nalidixic acid, the homologous double

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exchange colonies were selected in M22+ containing 10% (w/w) sucrose. For details,

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see the schematic showed by Porter et al. about the first and second recombination

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steps in chromosomal gene of Rb. Sphaeroides 23.Gene deletions and genomic

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integrations were verified by diagnostic PCR and DNA sequencing. All primers used

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for identification of the mutants are listed in Table S1.

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Shake-flask cultivation

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For shake-flask cultivation, wild type Rb. sphaeroides and subsequent recombinant

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strains were cultivated in rich medium (30 g/L glucose, 3 g/L corn steep liquor, 3 g/L

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sodium glutamate, 2.8 g/L NaCl, 3 g/L (NH4)2SO4, 3 g/L KH2PO4, 6.3 g/L MgSO4, 2

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g/L CaCO3, 1 mg/L thiamine hydrochloride, 15 mg/L biotin) according to Lu et al. 24. 7 / 29



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Shake-flask experiments were carried out in triplicate 250 mL Erlenmeyer flasks

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containing 125 mL fermentation medium and 2% inoculum. The cultures were

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incubated in the dark at 34 °C in a rotary shaking incubator at 150 rpm.

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Quantitative real time PCR (qPCR) of the related genes in the lycopene synthesis

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pathway

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To detect transcriptional levels of crtI4 and other related genes among the mutants and

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wild type, total RNA was isolated from the harvested cells by using the RNAiso plus

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Kit (Takara, Dalian, China) according to the manufacturer’s protocol. qPCR was

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performed using the SYBR tip green qPCR super mix Kit (Transgen, Beijing, China)

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according to the manufacturer’s protocol. The rpoZ gene, encoding a DNA-directed

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RNA polymerase, was chosen as the internal control gene to normalize the different

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samples 25. Four primer sets termed crtI3-RT-F/R,crtI4-RT-F/R, crtE-RT-F/R and dxs-

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RT-F/R for the lycopene synthesis key genes, crtI3, crtI4, crtE and dxs, were used for

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qPCR (PIKO REAL 96, Thermo Fisher Scientific, Waltham, Massachusetts) analysis.

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The primers are listed in Table S1. The relative expression of the genes was analyzed

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using the 2-△△ct method 26.

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Extraction and measurement of carotenoids

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The cells of Rb. sphaeroides were collected by centrifugation at 12,000 ×g for 2 min.

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The cell pellet was washed twice with distilled water and then dried at 80 °C to a

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constant weight to measure the DCW (10 mL fermentation broth). The lycopene was

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extracted (1mL fermentation broth) using the method of Martinez et al. 27 with some

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modifications. The cleaned cells were resuspended in 1 mL of acetone and incubated

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at 55 °C for 15 min in the dark with intermittent vortexing, then the sample was

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cooled in an ice bath, 1 mL hexane-water (1:1 v/v) was added, it was vortexed again

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and spun at 12,000 ×g for 2 min to gather carotenoids in upper layer hexane. Then the

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extraction filtered with a 0.45 µm-pore-size filter for HPLC (U3000, Thermo Fisher

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Scientific, Waltham, Massachusetts) analysis. The HPLC was equipped with a C18

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column, (4.6 mm × 250 mm) and UV/VIS detector set at 472 nm, and the mobile

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phase consisted of acetonitrile-methanol-isopropanol (80:15:5 v/v/v) with a flow rate

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of 1 mL/min at 30 °C 17. The lycopene standard (purity ≥ 96%) was purchased from

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Sigma-Aldrich. The lycopene of engineered strains were quantitative by HPLC, while

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the spheroidenone of wild type were calculated by the corresponding extinction

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coefficients. The extinction coefficient of spheroidenone was 122 mM−1 cm−1 which

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at 482 nm in 7:2 acetone : methanol (the extraction was dried in the dark under

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nitrogen, then re-dissolved in7:2 acetone : methanol) 28.

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Identification of lycopene

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The purification of the lycopene sample extracted from the engineered strain was

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conducted according to Marcos Rodríguez et al. 29. The lycopene was identified 9 / 29



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separately by fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic

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resonance (NMR) 30. The FTIR spectrum was measured using a FTIR Spectrometer

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TENSOR 27 (Bruker, Germany) in the spectral range 4000 to 400 cm-1 with a KBr

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window. 13C NMR (125 MHz) and 1H NMR (500 MHz) spectra were measured using

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an ASCEND 600 spectrometer (Bruker, Germany) in CDCl3 with tetramethylsilane as

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an internal standard.

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Statistical analysis

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All values are expressed as the mean ± standard deviation, and each value is the mean

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of separate experiments in triplicate. The statistical analysis of data and plots was

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performed using Origin software when necessary.

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Results

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Construction of a lycopene biosynthesis pathway in Rb. sphaeroides

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The carotenoid biosynthesis pathway and engineered strategies of Rb. sphaeroides are

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displayed in Figure 1. In the wild type Rb. sphaeroides, the carotenoids are

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synthesized via the glycolytic and MEP pathways, under photosynthetic conditions,

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the major end carotenoid was spheroidene, while under dark conditions, the major end

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carotenoid was spheroidenone. To construct the lycopene biosynthesis pathway in Rb.

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sphaeroides, an exogenous crtI4 needed to be introduced and crtC needed to be

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knocked out.

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The mechanism of different phytoene dehydrogenases that determine the

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dehydrogenation reaction have not yet been clarified. The function of a given

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dehydrogenase gene is only judged by identifying its products, thus we could only

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choose the optimal CrtI4 from reported bacteria 31. Since the wild type Rb.

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sphaeroides did not have CrtI4, we screened the reported bacteria that have native

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CrtI4 including Erwinia herbicola 16, R. rubrum 32, Rhodopseudomonas acidophila 33,

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Phaeospirillum molischianum and Rhodopseudomonas palustris 34 by protein

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homology analysis (similar comparison of additional CrtI sequences performed by

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Wang et al.32). Based on MAGE and NCBI BLAST, the sequence similarities of CrtI4

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sourced from the above mentioned bacteria with the native CrtI3 are 41%, 53%, 49%,

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50% and 49%, respectively. The CrtI4 from R. rubrum exhibited the highest similarity

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with 53% and their sequence details were analyzed by Vector NT1 (Fig. S1).

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Moreover, we found that the CrtI4 from R. rubrum exhibited good adaptation in Rb.

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sphaeroides 2.4.1 as its codon adaptation index was 0.66, the frequency of optimal

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codons was 64 and the GC content adjustment was 63.91 (analyzed by GenScript).

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Therefore, crtI4 from R. rubrum was chosen to replace the native crtI3. By integration

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of crtI4 and deletion of crtC in the wild type, the basal lycopene-producing strain was

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generated and termed RL1. The production of lycopene was noticeable as the cells

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turned color from purple (WT) to red (RL1). The strain was able to produce lycopene

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as the end-product, the lycopene content was 4.17 mg/g DCW and the lycopene

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concentration was 19.31 mg/L after 96 h of fermentation (Fig. 3). The biomass of 11 / 29



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RL1 decreased by 24% compared with the wild type (Fig. 3), which may be because

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the lycopene is somewhat toxic to microbial cells 8 and the carotenoid diversity was

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destroyed in the RL1 strain. We also have a Rb. sphaeroides mutant RL0 with only deletion of the crtC.

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Noticeably, the deletion of crtC made the cells to turn color from purple to yellow

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green as shown in Fig. S2, and resulted in 100% neurosporene accumulation as shown

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in HPLC chromatography of the total carotenoids extracted from RL0.

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Structure identification of lycopene

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We extracted and purified the lycopene sample from engineering strain RL1. First, it

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was identified by HPLC analysis based on the retention time compared with a

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standard sample (Fig. 2a). Additionally, FTIR and NMR analyses were performed to

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further confirm the structure of the end-product 35. From the FTIR data (Fig. 2b), the

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lycopene sample present in the extract showed a very strong swing vibration peak of

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E R–HC=CH–R at 958.57 cm-1, a very strong stretching peak of non-symmetric

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methyl (CH sp³) at 2914.29 cm-1, symmetric methylene (CH sp²) peaks at 2852.57 cm-

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1

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methylene (CH sp²) at 1442.68 cm-1, and a weak peak of stretching vibration of Z

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vinylene (R-CH=CH-R') at 3035.79 cm-1 corresponding to the stretching vibration

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peak of =C–H. There was a weak absorption peak between 730–665 cm-1, which was

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attributed to the absorption band of Z R–HC=CH–R.

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, a peak of methyl deformation (CH sp³) at 1375.17 cm-1, a medium bending peak of

The 13C NMR (151 MHz, CDCl3) experiment results (Fig. S3) for the lycopene

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showed peak as follows: δ 139.45 (s), 137.37 (s), 136.54 (s), 136.15 (s), 135.43 (s),

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135.15 (s), 132.67 (s), 131.72 (s), 131.58 (s), 130.09 (s), 126.58 (s), 125.76 (s), 12 / 29


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125.15 (s), 124.80 (s), 123.98 (s), 77.23 (s), 77.02 (s), 76.76 (d, J = 15.0 Hz), 40.25

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(s), 32.82 (s), 26.88 (s), 26.71 (s), 25.70 (s), 24.14 (s), 17.71 (s), 16.96 (s), 12.85 (d, J

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= 15.9 Hz), and 0.00 (s). The 1H NMR (600 MHz, CDCl3) spectra (Fig. S4) had the

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following peaks: δ 7.43 (s, 6H), 7.26 (s, 21H), 7.40 – 6.85 (m, 24H), 6.81 – 6.55 (m,

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39H), 6.55 – 6.35 (m, 31H), 6.34 (s, 8H), 6.30 – 6.24 (m, 31H), 6.18 (d, J = 11.4 Hz,

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20H), 5.95 (d, J = 10.7 Hz, 17H), 5.13 (d, J = 25.1 Hz, 16H), 2.26 – 2.01 (m, 79H),

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2.26 – 2.00 (m, 83H), 1.97 (s, 103H), 2.26 – 1.52 (m, 352H), 1.82 (s, 55H), 2.26 –

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1.23 (m, 364H), 2.26 – 1.18 (m, 366H), 1.70 (d, J = 17.2 Hz, 55H), 2.26 – 1.00 (m,

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372H), 1.65 – 0.42 (m, 85H), 1.53 – 0.42 (m, 27H), 0.86 (d, J = 21.1 Hz, 5H), and

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0.00 (s, 25H). From the HPLC retention time, FTIR and NMR data, the main

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ingredient in sample was identified as lycopene.

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Enhancing lycopene production by blocking the pentose phosphate pathway

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The channelization of carbon flux toward the target product is an effective metabolic

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engineering approach for improving target product biosynthesis. In Rb. sphaeroides,

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the glycolytic pathway supplies glyceraldehyde-3-phosphate (G3P) and pyruvate for

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lycopene biosynthesis. To maximize carbon flux towards the glycolytic pathway, the

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competitive pentose phosphate (PPP) pathway needs to be blocked. The gene zwf,

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encoding glucose-6-phosphate dehydrogenase, controls the entry of carbon into the

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PPP. Herein, we deleted zwf and generated a strain termed RL2. Compared with RL1,

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the deletion of zwf did not significantly affect biomass production, while the red color

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of the cells deepened and the lycopene content dramatically improved up to 7.82 mg/g

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DCW and the corresponding concentration was up to 36.91 mg/L (Fig. 3). The 13 / 29



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improvement of the lycopene content after zwf deletion was attributed to higher flux

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through tricarboxylic acid cycle to replace anabolic reducing equivalents normally

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provided by oxidative pentose phosphate pathway 19, and saved more carbon

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metabolic flux to glycolytic pathway which relate to the increase of precursor

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availability.

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In the central carbon metabolic pathway, Zwf, Pgi (glucosephosphate isomerase)

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and GdhA (glutamate dehydrogenase) are identified as the key enzymes. Specifically,

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Zhou et al. demonstrated that Zwf played the most significant role in central

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metabolic flux 19. Here, the knockout of zwf resulted in an elevation of the lycopene

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content by 88%, which proved that zwf is also an effective target for lycopene

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improvement in Rb. sphaeroides.

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Boosting lycopene biosynthesis by integration of the dxs gene

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Except for central carbon metabolism, metabolic engineering of the MEP pathway for

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terpenoid precursor overproduction is also an important strategy to improve lycopene

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production 36. The first reaction of the MEP pathway is catalyzed by Dxs, which links

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central carbon metabolism to lycopene metabolism. In this study, dxs was integrated

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in the zwf site to generate the strain termed RL3. Compared with the RL2 strain, the

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lycopene content was elevated by 18% and the biomass increased by 15% in RL3.

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The lycopene content increased to 9.26 mg/g DCW and the corresponding 14 / 29


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concentration was 50.10 mg/L (Fig. 3).This improvement was mainly attributed to

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routing the carbon flux into the pool of the MEP pathway resulting in the elevated

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amount of lycopene.

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In the MEP pathway, the Dxs, Idi (isopentenyl diphosphate isomerase), and Dxr

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(1-deoxy-D-xylulose 5-phosphate reductoisomerase) are commonly recognized as the

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rate-limiting enzymes for the isoprenoid flux delivery 37. dxs serves as the first gene

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of the MEP pathway, and determines it’s flow. Kim and Keasling have demonstrated

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that dxs is one of the most influential genes involved in the precursor supply

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compared with other isoprenoid biosynthesis genes 17. Here, the integration of dxs in

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the zwf site resulted in an improvement of the lycopene content by 122% when

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compared with the basal lycopene-producing strain RL1. Both zwf deletion and dxs

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integration contributed to the channelization of carbon flow for precursor

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overproduction, which provides a promising strategy and theoretical basis for

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increasing the synthesis of lycopene in Rb. sphaeroides or other photosynthetic

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bacteria.

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Transcriptional level of related genes in the lycopene biosynthesis pathway

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To clarify the regulatory mechanism of carotenogenesis in recombination strains, the

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transcription levels of crtE, crtI3, crtI4, and dxs were separately studied in wild type,

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RL1, RL2 and RL3 strain. Compared to the reference gene rpoZ, the expression level 15 / 29



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of native crtI3 in WT was 1.01, while the expression level of crtI4 in RL1 was 0.57

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(Fig. 4). This results account for the lower content of carotenoid in RL1 than WT.

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Compared with RL1, the deletion of zwf led to increasing the expression level of crtI4

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in RL2 by 43% and this improvement contributed to an increase in lycopene content

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of 88%. This implied that the expression level of crtI4 was positively correlated with

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the synthesis of lycopene. Due to the native crtI3 promoter was retained by crtI4

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seamless replacement, the increased crtI4 expression level also should regulated by

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the promoter of puc and puf operon as the Lang et al. have reported 38. Compared with

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RL2, the integration of dxs give rise to the expression level of dxs increased by 53%

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in RL3 (Fig. 4), which also account for the improvement of lycopene content by 18%.

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The integration of dxs also led to the return of the expression level of the secondary

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metabolic gene crtE (geranylgeranyl pyrophosphate synthase) to the normal level (Fig.

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4), which also contributed to the promotion of carotenogenic ﬂux. This phenomenon

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indicated the carotenogenic genes are co-regulated and that the genetic manipulation

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of only one gene will induce the entire pathway.

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Shake-flask fermentation curve of strain RL3

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To further understand the lycopene production of Rb. sphaeroides, the cell growth,

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glucose consumption and lycopene accumulation were monitored every 24 h in the

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RL3 and wild type separately. To verify the stability of the RL3 engineered strain and 16 / 29


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ensure the accuracy of the fermentation data, we repeated four batch fermentation

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experiments and the results showed that the fermentation was reproducible. In the

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early stage of fermentation (0–48 h), the fermentation broth was milky yellow then

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gradually changed to pink (48–72 h), red (72–96 h), and then deep red (96–168 h).

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One batch was chosen for further analysis (shown in Fig. 5a). After 168 hours of

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fermentation, the biomass reached 6.4 g/L, the glucose concentration decreased from

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30.0 g/L to 15.33 g/L, the lycopene concentration reached 66.05 mg/L and the

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lycopene content was 10.32 mg/g DCW. In fermentation of WT (Fig. 5b), the growth

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and consumption of glucose were the similar to RL3. This suggested there no effect

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on growth and glucose’s consumption of Rb. sphaeroides by genetic manipulation.

321

Discussion

322

At present, microbial producing lycopene mostly focused on employing natural

323

carotenogenic microorganisms: B. trispora, and metabolically engineered non-

324

carotenogenic microorganisms: E. coli, S. cerevisiae. Until now, the highest lycopene

325

content had achieved up to 448 mg/g DCW in engineered E. coli 39, B. trispora

326

realized 83.2 mg/g DCW 40, and S. cerevisiae realized 55.56 mg/g DCW 41.

327

Although these accesses provide a good yield, but the above mentioned high

328

level production have used complicated metabolic engineering techniques 42.

329

Compared with these chassis, phototrophic bacteria as a new platform for lycopene

17 / 29



330

production have natural advantages. Phototrophic bacteria naturally produce a range

331

of carotenoids that function by protecting the photosynthetic apparatus against

332

harmful radicals arising from both light and oxygen 10. Rb. sphaeroides, a model

333

phototrophic bacterium, exhibits great superiority as a cell factory for carotenoids

334

production. Herein, we constructed a lycopene-producing strain in Rb. sphaeroides by

335

manipulating only four genes and all the mutations were the seamlessly replaced and

336

deleted and use native promoters. Thus, not only avoided the increasing metabolic

337

cost of antibiotic resistance genes but also ensured the strains had a strong stability.

338

Rb. sphaeroides was demonstrated to be a competitive producer of lycopene

339

among phototrophic bacteria

340

That Rb. sphaeroides produces lycopene was determined by the type of phytoene

341

dehydrogenase. In 2002, Garcia-Asua et al. used a heterologous CrtI4 from Erwinia

342

herbicola (now called Pantoea agglomerans) to synthesize lycopene in Rb.

343

sphaeroides to study its function in the photosystem, resulting in an increase in the

344

proportion of lycopene among carotenoids to 93% 16. Wang and Liao by directed

345

evolution CrtI3 to generate lycopene in Rb. sphaeroides, and increased the percent of

346

lycopene to 90% among the carotenoids 43. A basal lycopene-producing strain was

347

constructed in Rb. sphaeroides by manipulating only two genes, deleting crtC and

348

substituting native crtI3 with crtI4 from R. rubrum, resulting in an increase in the 18 / 29


Page 18 of 35

Page 19 of 35


349

lycopene proportion up to 96%. This basal strain produced 4.17 mg/g DCW of

350

lycopene from glucose. Similarly, in another photosynthesis bacterium, R. rubrum,

351

the deletion of crtC and crtD allowed for only 2 mg/g DCW lycopene production

352

from succinate and fructose 32. What’s more, in other photosynthetic bacteria, the

353

research primarily focused on total carotenoids production via optimization of the

354

cultivation conditions. The final engineered strain RL3 produced 10.32 mg/g DCW of

355

lycopene, to the best of our knowledge, this is the highest reported yield of lycopene

356

in photosynthetic microorganisms. In general, this study demonstrated that Rb.

357

sphaeroides is a competitive producer of lycopene among phototrophic bacteria.

358

Further improvement strategies in engineered Rb. sphaeroides

359

In the shake-flask cultivation, the glucose was not exhausted, indicating that the

360

glucose concentration was too high, and as Alper et al. suggested 44, carotenoid

361

production was significantly repressed in the glucose-containing medium. This

362

phenomenon suggested that if a more suitable carbon source was chosen for the

363

fermentation of engineered Rb. sphaeroides, there is still potential for the yield to

364

further improve. Additionally, cofactor availability is an efficient strategy to increase

365

biosynthesis. Thus, Rb. sphaeroides is attractive as a host as it has the ability to utilize

366

sunlight and carbon dioxide, both economical resources 45. It is a remarkable fact that

367

the biosynthesis of 1 mol of lycopene requires 16 mol of NAD(P)H, therefore Rb. 19 / 29



368

sphaeroides has specific advantages for lycopene production because of its capable of

369

regenerating co-factors through photosynthesis 46. We have done preliminary

370

experiments of producing lycopene under light condition by RL3 and WT (shown in

371

supplementary Fig. S5), the results indicate that the lycopene synthesis under light

372

condition is complexity, so it is another challenge in need of resolution. It is also

373

anticipated that microbial production of lycopene derived from solar energy and

374

carbon dioxide will be realized in the near future, and our lab is committed to

375

realizing this vision.

376

Supporting Information

377

The Supporting Information is available free of charge on the ACS Publications

378

website at DOI:. Table S1 showing primers used in this study. Figure S1 showing the

379

sequence comparison details of phytoene desaturase (CrtI). Figure S2 and Figure S3

380

showing the 13C NMR (125 MHz) and 1H NMR (500MHz) spectrum of the lycopene

381

sample extracted from RL1, respectively.

382

Funding

383

This research was supported by the fundamental research funds for the central

384

universities (Project No.2016CSY019), central college fund of special support project

385

of China (GK GK201706010), the national key research and development program of

386

China (2017YFD0400702).

20 / 29


Page 20 of 35

Page 21 of 35


387

Conflict of interest

388

The authors declare no competing financial interest.

389

Abbreviations

390

CrtC, neurosporene hydroxylase; CrtI3, 3-step dehydrogenase; CrtI4, 4-step

391

dehydrogenase; DCW, dry cell weight; Dxs, The 1-deoxy-D-xylulose-5-phosphate

392

synthase; FTIR, fourier-transform infrared spectroscopy; MEP, the methylerythritol

393

phosphate; NMR, nuclear magnetic resonance; PPP, pentose phosphate; Zwf, glucose-

394

6-phosphate dehydrogenase.

395

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in

phototrophic


538

Figure Legends

539

Fig. 1 The simplified lycopene synthesis pathway in engineered Rb. sphaeroides.

540

CrtI3: three-step dehydrogenase (from Rhodobacter sphaeroides), CrtI4: four-step

541

dehydrogenase (from Rhodospirillum rubrum), CrtC: neurosporene hydroxylase, Zwf:

542

glucose-6-phosphate dehydrogenase, Dxs: 1-deoxy-D-xylulose-5-phosphate synthase.

543

Fig. 2 Identification of lycopene. a: HPLC chromatogram of the lycopene standard

544

and carotenoids extracted from RL1 and WT. The lycopene structure is shown above

545

the lycopene standard peak. b: FITR of the lycopene sample extracted from RL1.

546

Fig. 3 The carotenoids content, biomass, concentration and OD600 of wild type and

547

three engineered lycopene strains after 96 h of fermentation. The corresponding

548

lycopene contents are shown in the table below. Error bars represent the SD from

549

three independent biological replicate experiments.

550

Fig. 4 Relative expression levels of crtE, crtI3, crtI4 and dxs genes in different strains.

551

Data represent the average of three replicates and error bars represent the standard

552

deviation.

553

Fig. 5 The fermentation curves of carotenoids content, biomass, OD600 and glucose

554

consumption in WT and RL3. Error bars represent the SD from three independent

555

biological replicate experiments.

556

28 / 29


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Page 29 of 35


557

Table 1 Strains and plasmids used in this study Strains

Description

Sources

endA1 hsdR17 [r-m+] supE44 thi-1 recA1 gyrA E. coli DH5α

[NalR] relA relA1 ∆[lacZYA-argF] U169 deoR

Novagen

[Ø80∆ (LacZ) M15] Thi endA recA hsdR with RP4-2-Tc::MuE. coli S17-1

Novagen Km::Tn7 integrated in chromosome

Rhodobacter sphaeroides

Wild type; ATH 2.4.1

Our lab

Rhodospirillum rubrum

Wild type UR2

Our lab

RL1

△crtI3::crtI4△crtC

This study

RL2

△crtI3::crtI4△crtC△zwf

This study

RL3

△crtI3::crtI4△crtC△zwf::dxs

This study

Plasmids

Used for

Sources

pK18mobsacB

Suicide vector, sacB (sucrose sensitivity); Kmr

Takara

pK18-△crtI3::crtI4

Replacing native crtI3 with crtI4; Kmr

This study

pK18-△crtC

Knocking out crtC; Kmr

This study

pK18-△zwf

Knocking out zwf; Kmr

This study

pK18-△zwf::dxs

Replacing zwf with dxs; Kmr

This study

558 559

29 / 29



Fig. 1 The simplified lycopene synthesis pathway in engineered Rb. sphaeroides. CrtI3: three-step dehydrogenase (from Rhodobacter sphaeroides), CrtI4: four-step dehydrogenase (from Rhodospirillum rubrum), CrtC: neurosporene hydroxylase, Zwf: glucose-6-phosphate dehydrogenase, Dxs: 1-deoxy-Dxylulose-5-phosphate synthase. 151x96mm (300 x 300 DPI)


Page 30 of 35

Page 31 of 35


Fig. 2 Identification of lycopene. a: HPLC chromatogram of the lycopene standard and carotenoids extracted from RL1 and WT. The lycopene structure is shown above the lycopene standard peak. b: FITR of the lycopene sample extracted from RL1. 127x39mm (300 x 300 DPI)



Fig. 3 The carotenoids content, biomass, concentration and OD600 of wild type and three engineered lycopene strains after 96 h of fermentation. The corresponding lycopene contents are shown in the table below. Error bars represent the SD from three independent biological replicate experiments. 243x83mm (300 x 300 DPI)


Page 32 of 35

Page 33 of 35


Fig. 4 Relative expression levels of crtE, crtI3, crtI4 and dxs genes in different strains. Data represent the average of three replicates and error bars represent the standard deviation. 208x159mm (300 x 300 DPI)



Fig. 5 The fermentation curves of carotenoids content, biomass, OD600 and glucose consumption in WT and RL3. Error bars represent the SD from three independent biological replicate experiments. 290x104mm (300 x 300 DPI)


Page 34 of 35

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TOC graphic 82x44mm (300 x 300 DPI)


Metabolic Redesign of Rhodobacter sphaeroides for Lycopene

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