Deinococcus wulumuqiensis R12 is a red-pigmented extremophilic microorganism with powerful antioxidant proper- ties that was isolated from radiation-contaminated soil in Xinjiang Uyghur Autonomous Region of China. The key carotenoid biosynthesis genes, crtE, crtB and crtI, which are related to the cells’ antioxidant defense, were identified in the sequenced genome of R12 and analyzed. In order to improve the carotenoid yield in engineered Escherichia coli, the origin of carotenoid biosynthesis genes was discussed, and a strain containing the R12 carotenoid bio- synthesis genes was constructed to produce lycopene, an important intermediate in carotenoid metabolism. The gene order and fermentation conditions, including the culture medium, temperature, and light, were optimized to obtain a genetically engineered strain with a high lycopene production capacity. The highest lycopene content was −1 688 mg L in strain IEB, which corresponds to a 2.2-fold improvement over the original recombinant strain EBI. Keywords: Lycopene, Escherichia coli, Gene regulation, Fermentation optimization Introduction Escherichia coli, Saccharomyces cerevisiae, Candida uti- Lycopene is a representative molecule from the carot- lis, or Yarrowia lipolytica (Hernández-Almanza et al. enoid family, and is one of the strongest antioxidants 2016; Mantzouridou and Tsimidou 2008; Miura et al. known to date. Due to its physiological effects (e.g. 1998). A new species with powerful antioxidant capacity, immune enhancement, free radical scavenging), lyco- Deinococcus wulumuqiensis R12, was screened from an pene is widely used in various fields, such as medicine, irradiated area in Xinjiang province (Wang et al. 2010). It food and cosmetics (Moise et al. 2013; Ciriminna et al. appears red to the unaided eye because of its production 2016). Lycopene production by microbial fermentation of carotenoids, which is one of the major mechanisms has attracted much attention in recent years because of of its radiation resistance. Due to this, the radiation- the identification of biosynthetic genes and the discovery resistant R12 strain can be used as a new platform for of new highly productive pigment-producing strains. The carotenoid synthesis, as well as a model for research on strains that are used to produce lycopene mainly include the biological adaptations of extremely radioresistant microbes that can synthesize lycopene naturally, such as bacteria. Blakeslea trispora, Erwinia herbicola, Rhodotorula genus, There are known two lycopene-synthesis pathways in or Dunaliella salina, and engineered microbes, such as microorganisms. One is the mevalonate (MVA) path- way, which is present in all known eukaryotic cells *Correspondence: firstname.lastname@example.org; email@example.com and the cytoplasm and mitochondria of plants, and School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, the other is the 2-C-methyl-d -erythritol-4-phosphate Jiangsu Province, China (MEP) pathway present in bacteria, other prokaryotes College of Food Science and Light Industry, Nanjing Tech University, Nanjing, Jiangsu Province, China and the plastids of plants (Hernández-Almanza et al. Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Xu et al. AMB Expr (2018) 8:94 Page 2 of 13 2016). Lycopene is a typical product of a multi-enzyme Alper et al. 2005; Yoon et al. 2007). Some strategies catalytic pathway, in which isopentenyl pyrophos- have improved lycopene production by regulating the phate (IPP), dimethylallyl pyrophosphate (DMAPP) expression of key genes, gene knockouts, changing the and farnesyl pyrophosphate (FPP) are synthesized external conditions, and adding exogenous substances by 8 sequential enzymes in the MEP pathway, after (Yan et al. 2013; Alper et al. 2005; Yoon et al. 2007; Kim which they are converted to lycopene by the three key et al. 2011; Bhosale 2004; Roukas 2015; Zhu et al. 2015; enzymes geranylgeranyl diphosphate synthase (encoded Matthäus et al. 2014; Arayagaray et al. 2012; Bahieldin by crtE), phytoene synthase (encoded by crtB), and phy- et al. 2014). In these genetic engineering strategies, the toene desaturase (encoded by crtI) (Fig. 1). Lycopene co-expression of key lycopene synthesis genes in hosts can then be converted into a variety of carotenoids and constitutes the traditional approach, which may lead to derivatives in different organisms through modification an imbalance of metabolic fluxes that negatively affects reactions such as cyclizations, oxygenations and dehy- the product yield. It is therefore imperative to preserve drogenations, which makes it one of the most impor- the balance of metabolic fluxes in these multi-gene tant intermediates in the carotenoid family. With the expression systems, which requires intensive study. development of metabolic engineering and synthetic In this study, lycopene biosynthesis genes from the biology, lycopene production by microbial fermenta- newly discovered species Deinococcus wulumuqien- tion has gained increasing attention from researchers sis R12 were identified, analyzed, and integrated into a due to its advantages of lower potential cost and sim- polycistronic plasmid for expression in Escherichia coli. pler, safer processes. The lycopene biosynthesis genes Lycopene production of the recombinant strain was from various microorganisms, such as Erwinia uredo- investigated in different culture media, and under differ - vora, Erwinia herbicola, Pantoea ananatis, Pantoea ent temperature and light conditions. Finally, plasmids agglomerans, and Brevibacterium linens, have been with the lycopene biosynthesis genes crtE, crtB, and crtI co-expressed in recombinant strains (Yan et al. 2013; arranged in different order were constructed to study Fig. 1 The biosynthesis pathways of lycopene and other carotenoids. The MVA pathway is found in eukaryotic cells, the cytoplasm and mitochondria of plants. The MEP pathway is found in bacteria, other prokaryotes and plastids in plants. The carotenoid synthesis pathway in Deinococcus radiodurans R1 was marked with red arrows. G3P glyceraldehyde 3-phosphate, DXP 1-deoxy-d -xylulose-5-phosphate, MEP 2-C-methyl-d -erythritol-4-phosphate, DMAPP dimethylallyl diphosphate, IPP isopentenyl diphosphate, HMG-CoA 3-hydroxy-3-methyl glutaryl coenzyme A, MVA mevalonate, FPP farnesyl diphosphate, GGPP geranylgeranyl diphosphate Xu et al. AMB Expr (2018) 8:94 Page 3 of 13 the effect of gene order, which is related to the individual was used to inoculate 50 mL of medium and incubated genes’ translation efficiency, on the lycopene yield. at 37 °C and 200 rpm for 3 h. The cultures were then fer - mented with or without isopropyl-β-d-thiogalactoside Materials and methods (IPTG, 0–1 mM) under different conditions. Where −1 Bacterial strains, plasmids, and growth conditions appropriate, 100 mg L of ampicillin was added to pro- All bacterial strains and plasmids used in this study are mote plasmid retention. Cultivation was conducted in listed in Table 1. E. coli DH5α and E. coli BL21 (DE3) cells the dark in biological triplicates. To determine the dry were used for cloning and gene expression, respectively. cell weight (DCW), 1 mL of the sample was centrifuged D. wulumuqiensis R12 (CGMCC 1.8884 ) (Wang et al. (13,000×g, 5 min), washed twice with double-distilled −1 2010) was grown in TGY medium (10 g L of tryptone, water, centrifuged again and dried at 100 °C until con- −1 −1 1 g L of glucose, and 5 g L of yeast extract) at 30 °C. stant weight. Recombinant E. coli cells were grown at 37 °C in Luria– −1 −1 Bertani (LB) medium (10 g L of tryptone, 5 g L Genome sequencing and bioinformatics analysis −1 of yeast extract, and 10 g L of NaCl), 2×YT medium of carotenoid‑biosynthesis genes from D. wulumuqiensis −1 −1 (16 g L of tryptone, 10 g L of yeast extract, and R12 −1 5 g L of NaCl), 2× YT + G medium (2× YT medium The genomic DNA of Deinococcus wulumuqiensis R12 −1 with 10, 20, 40, 60, 80, or 100 g L glycerol), or synthetic was isolated using a genomic DNA extraction kit (Takara, −1 −1 medium (SM) [10 g L of glycerol, 10 g L of glucose, China). The draft genome sequence of strain R12 was −1 −1 −1 7.5 g L of L-arabinose; 11.2 g L of KH PO , 3 g L of obtained using the Illumina MiSeq platform, which 2 4 −1 −1 (NH ) HPO , 0.3 g L of NaCl, 1 g L of MgSO ∙7H O, was performed by BGI Tech Solutions Co., Ltd., China, 4 2 4 4 2 −1 −1 −1 1.1 g L of leucine, 0.7 g L of isoleucine, 0.4 g L of using a paired-end library. This whole-genome shotgun −1 −1 −1 valine, 1.5 g L of threonine, 2 g L of lysine, 3.3 g L sequence has been deposited with GenBank under the −1 −1 of phenylalanine, 2.2 g L of glutamine, and 3.3 g L of Accession No. APCS00000000 (http://www.ncbi.nlm.nih. methionine] (Kim et al. 2011). For lycopene production, gov/nucco re/APCS0 00000 00). The functional annota - a single colony was used to inoculate 50 mL of medium tion of proteins was conducted using different databases, in a 250 mL flask, which was then incubated at 37 °C and including Gene Ontology (GO, Version:1.419) (Ash- 200 rpm for 16 h. Subsequently, 3 mL of the pre-culture burner et al. 2000), Cluster of Orthologous Groups of Table 1 Bacterial strains and plasmids used in this study Plasmid Relevant properties Source pET-22b Amp , T7 promoter Invitrogen pET-E Amp , carrying the crtE gene from D. wulumuqiensis R12 This study pET-EB Amp , carrying the crtE and crtB genes from D. wulumuqiensis R12 This study pET-EBI Amp , carrying the crtE, crtB and crtI genes from D. wulumuqiensis R12 This study pET-EIB Amp , carrying the crtE, crtI and crtB genes from D. wulumuqiensis R12 This study pET-BEI Amp , carrying the crtB, crtE and crtI genes from D. wulumuqiensis R12 This study pET-BIE Amp , carrying the crtB, crtI and crtE genes from D. wulumuqiensis R12 This study pET-IEB Amp , carrying the crtI, crtE and crtB genes from D. wulumuqiensis R12 This study pET-IBE Amp , carrying the crtI, crtB and crtE genes from D. wulumuqiensis R12 This study Strains Deinococcus wulu- Aerobic, Gram-positive, non-spore-forming, nonmotile, tetrad-forming coccus; forming reddish-orange, circu- ( Wang et al. 2010) muqiensis R12 lar, opaque colonies (approx. 1.8–3.8 mm in diameter) after incubation on TGY medium for 14 days at 37 °C E. coli DH5α deoR endA1 gyrA96 hsdR17 (rK− -mK+) recA1 relA1 supE44 thi-1 Δ (lacZYA-argF) U169 Φ80lacZ ΔM15 F -λ- Vazyme E. coli BL21(DE3) F− ompThsdS (rB− mB−) gal dcm (DE3) Vazyme EDWe Amp , E. coli BL21(DE3) containing the plasmid pET-22b This study EBI Amp , E. coli BL21(DE3) containing the plasmid pET-EBI This study EIB Amp ,E. coli BL21(DE3) containing the plasmid pET-EIB This study BEI Amp , E. coli BL21(DE3) containing the plasmid pET-BEI This study BIE Amp , E. coli BL21(DE3) containing the plasmid pET-BIE This study IEB Amp , E. coli BL21(DE3) containing the plasmid pET-IEB This study IBE Amp , E. coli BL21(DE3) containing the plasmid pET-IBE This study Xu et al. AMB Expr (2018) 8:94 Page 4 of 13 proteins (COG, Version:20090331) (Tatusov et al. 2003), Table 2 Primers used in this work Kyoto Encyclopedia of Genes and Genomes (KEGG, Ver- GenesPrimer sequence Restriction sion:59) (Kanehisa et al. 2006), and the NR database in enzyme site GenBank. The secondary metabolite gene clusters were predicted using the antiSMASH (Antibiotics and Sec- crtE1 F: 5′- GATC CAT ATG CGT CCC GAA CTG -3′ NdeI ondary Metabolite Analysis Shell) online tool (http:// R: 5′- CTT GAA TTC CTT CTC CCG CGT CGC -3′ EcoRI stoth ard.afns.ualbe rta.ca/cgvie w_serve r/) (Weber et al. crtB1 F: 5′- CCG GAA TTC GTG ACG GAA TTT TCGCC -3′ EcoRI 2015). The carotenoid biosynthesis genes from R12 was HindIII R: 5′- CCC AAG CTT GCC GTG GGC GGC GTC -3′ blasted with the type strain Deinococcus radiodurans crtI1 F: 5′-CCC AAG CTT ATG ACA TCC CCT CTT CCC TG -3′ HindIII R1 in GenBank. Multiple sequence alignment was con- XhoI R: 5′- CCG CTC GAG TCA GCG CCG GAT GTCG -3′ ducted by Vector NTI (Version: 11.5.1). The enzymes crtI2 F: 5′- CCG GAA TTC ATG ACA TCC CCT CTT CCC TG -3′ EcoRI in carotenoid biosynthesis encoded by these genes were R: 5′- CCC AAG CTT GCG CCG GAT GTC G -3′ HindIII analyzed by bioinformatics. Theoretical isoelectric point crtB2 HindIII F: 5′-CCC AAG CTT GTG ACG GAA TTT TCGCC -3′ and molecular weight was calculated by Compute pI/Mw R: 5′- CCG CTC GAG TCA GCC GTG GGC GGC GTC -3′ XhoI tool (http://us.expas y.org/tools /pi_tool.html). SignalP crtB3 NdeI F: 5′- GATC CAT ATG GTG ACG GAA TTT TCGCC -3′ (http://www.cbs.dtu.dk/servi ces/Signa lP-1.1/) was used R: 5′- CTT GAA TTC GCC GTG GGC GGC GTC -3′ EcoRI to predict the signal peptide of these enzymes. Trans- crtE2 F: 5′- CCG GAA TTC ATG CGT CCC GAA CTG -3′ EcoRI membrane prediction program TMHMM (http://www. R: 5′- CCC AAG CTT CTT CTC CCG CGT CGC -3′ HindIII cbs.dtu.dk/servi ces/TMHMM -2.0/) was applied to iden- crtE3 F: 5′- CCC AAG CTT ATG CGT CCC GAA CTG -3′ HindIII tify transmembrane regions. XhoI R: 5′- CCG CTC GAG TCA CTT CTC CCG CGT CGC -3′ crtI3 F: 5′- GATC CAT ATG ATG ACA TCC CCT CTT CCC TG -3′ NdeI DNA manipulation and plasmid construction EcoRI R: 5′- CTT GAA TTC GCG CCG GAT GTC G -3′ Fragments encoding crtE, crtB, and crtI were individually amplified from the genomic DNA of D. wulumuqiensis Restriction sites are underlined R12 using the primers listed in Table 2. The termination codon TAA of crtB and crtI was removed using appro- supernatants were used for HPLC analysis. All extraction priately designed primers. The crtE PCR fragment was operations were conducted in the dark. digested with NdeI and EcoRI, purified, and ligated into For HPLC analysis, 20 µL of each supernatant was ana- the plasmid pET-22b to construct pET-E. The plasmid lyzed using a Venusil XBP C18 column (4.6 × 150 mm, pET-EB was constructed by digesting the crtB fragment 5 µm; Agela Technologies, USA), kept at 30 °C, and eluted with EcoRI and HindIII, purifying, and ligating into plas- with a mobile phase comprising 80% acetone, 15% meth- mid pET-E. The fragment crtI was digested with Hin dIII −1 anol, and 5% isopropanol at a flow rate of 1 mL min for and XhoI, purified, and ligated into plasmid pET-EB to 40 min. The absorption of the acetone-extracted pigment construct pET-EBI. The fragments crtE1, crtE2, crtE3, mixture was detected at 472 nm. Commercial lycopene crtB1, crtB2, crtB3, crtI1, crtI2 and crtI3 with different (Sigma-Aldrich, USA) dissolved in acetone was used as a restriction enzyme sites were amplified using the cor - positive control. All results represent the means ± stand- responding primers listed in Table 2, and cloned into ard deviations of three independent experiments. pET22b to form five recombinant plasmids with a differ - ent orders of the three genes, pET-EIB, pET-BEI, pET- Nucleotide sequences BIE, pET-IBE, pET-IEB, in a similar manner as pET-EBI The nucleotide sequences of crtE, crtB and crtI from D. (Fig. 2). Each plasmid was sequenced after each gene liga- wulumuqiensis R12 were submitted to the GenBank data- tion, and transferred into E. coli BL21 (DE3), resulting in base with Accession Numbers KP319019, KP319020, and the strains EBI, EIB, BEI, BIE, IBE, and IEB, respectively. KP319021, respectively. pET-22b was introduce into E. coli BL21 (DE3) to form EDWe, which was used as the negative control. Results Identification of a carotenoid biosynthetic gene cluster Isolation of carotenoids and analytical methods from the genome of D. wulumuqiensis R12 After cultivation, the cells from 10 mL of culture broth Deinococcus wulumuqiensis R12 was isolated from radi- were harvested by centrifugation at 13,000×g and 4 °C ation-contaminated soils found in Xinjiang Province, for 5 min. The resulting cell pellets were collected, China, and the whole genome of R12 was sequenced washed once with double-distilled water, resuspended in and analyzed in a previous study (Xu et al. 2013). Func- acetone and incubated at 55 °C for 15 min, followed by tional annotation was completed by blasting predicted renewed centrifugation (13,000×g, 25 °C, 10 min). The Xu et al. AMB Expr (2018) 8:94 Page 5 of 13 Fig. 2 Construction of recombinant plasmids with different gene order of crtE, crtB and crtI. The fragments crtE1, crtE2, crtE3, crtB1, crtB2, crtB3, crtI1, crtI2 and crtI3 with different restriction enzyme sites were amplified and cloned into pET22b to form six recombinant plasmids with different gene orders of crtE, crtB and crtI. The termination codon TAA of the first and second genes was removed genes against the GO, COG and KEGG databases (Addi- marked with red in Fig. 1. The key genes for the syn - tional file 1: Figure S1). During the annotation, we found thesis of carotenoids in R12 were identified by BLAST a number of genes related to metabolic pathways of comparison against the genome of R1 (Anderson et al. secondary metabolites and terpenoids. There were 56 1956). The results of bioinformatic analysis of these genes genes related to the secondary metabolites biosynthesis, and enzymes were shown in Additional file 1: Table S1. transport and catabolism according to the gene func- Seven corresponding ORFs in the R12 genome, orf01490, tion annotation of the COG database. According to the orf00123, orf00124, orf01641, orf02322, orf03006, and KEGG database annotation results, 63 genes were found orf02323, showed 85.5, 86.3, 86.8, 82.2, 78.7, 81.0 and to be related to the metabolism of terpenoids and pol- 90.3% sequence identity to DR1395 (crtE, encoding gera- yketides (Additional file 1: Figure S1). In addition, the nylgeranyl diphosphate synthase), DR0862 (crtB, encod- terpenoid pathway, carotenoid biosynthesis pathway, ing phytoene synthase), DR0861 (crtI, encoding phytoene and related genes in the R12 genome were annotated via desaturase), DR0801 (crtLm, encoding lycopene cyclase), the KEGG pathway database. Using antiSMASH, 19 sec- DR0091 (cruF, encoding carotenoid 1′2′-hydratase), ondary metabolic gene clusters were predicted, of which DR2250 (crtD, encoding C-3′4′ desaturase) and DR0093 cluster 2 and cluster 13 were associated with the terpene (crtO, encoding carotene ketolase) of R1, respectively. pathway. The similarity of these two gene clusters, which Alignment of amino acid sequences showed 85.2, 81.9, were closest to that of Deinococcus radiodurans R1, the 90.9, 81.0, 77.0, 83.7, and 93.9% sequence identity to the type strain of radiation resistant microorganisms, was 31 corresponding proteins of R1. The isoelectric points of and 26%, respectively (Additional file 1: Figure S2). These the proteins were between 5 and 10. The C-3′4′ desatu - results indicated that the R12 genome indeed contains rase encoded by orf03006 had a signal peptide, and carot- genes related to the synthesis of terpenes. However, the enoid 1′2′-hydratase encoded by orf02322 had seven orientation and distributions of these homologous genes transmembrane domains. The other corresponding pro - were distinctly different from those of Deinococcus radio - teins had no signal peptide or transmembrane domains, durans R1. suggesting that they were intracellular enzymes. The ori - There were seven key genes involved in the produc - entation and distribution of the carotenoid biosynthesis tion of carotenoids in Deinococcus radiodurans R1, and genes in the R12 draft genome sequence was illustrated the key genes and carotenoid synthesis pathway were by arrows, compared to those in the whole-genome Xu et al. AMB Expr (2018) 8:94 Page 6 of 13 Lycopene production in E. coli using carotenoid genes sequence of R1 (GenBank No. NC001263) (Additional from D. wulumuqiensis R12 file 1: Figure S3). The carotenoid biosynthesis genes did In carotenoid synthesis, lycopene is formed from FPP not constitute a gene cluster in the genomes of these two by three key enzymes, which are encoded by crtE, strains, and were distributed in different loci. Although crtB and crtI (Fig. 1). These three genes from D. wulu - the genes from R12 were distributed to different scaffolds muqiensis R12 were assembled to form pET-EBI, and of the genome, their orientation and order were the same introduced into Escherichia coli BL21 (DE3). Protein as in the genome of R1. expression was induced using IPTG. The acetone super - However, when BLAST analysis of these carotenoids natants from the EBI strain were separated for 30 min genes was carried out in the NCBI nucleotide database by HPLC, and no lycopene was found in the control (BLASTN 2.8.0+), there were fewer genes similar to strain EDWe carrying the empty vector pET-22b. Col- those in the genome of R12. Firstly, there were less than onies of the EBI strain appeared red and the specific 20 genes similar to the key carotenoid synthesis genes peak of lycopene was identified by comparing it with of R12, with a low gene similarity in more than 30% of a commercially available authentic lycopene standard. the cases. In addition, most of these sequences only had −1 The strain produced a lycopene content of 312 mg L , sequence-based genomic annotations without experi- proving that crtE, crtB and crtI are indeed the lycopene mental verification of gene function. Secondly, the strains synthesis genes of D. wulumuqiensis R12. The effect with genes similar to those from R12 were grouped in the of different IPTG concentrations was investigated in genera Deinococcus and Thermus, as well as new genera the recombinant strain EBI (Fig. 3). The lycopene yield discovered in recent years. There were obvious differ - reached the highest value at 42 h, while the biomass ences between these 7 carotenoid biosynthesis genes and reached the maximum at 30–36 h. With the increase similar key genes in other Deinococcus species, owing to of IPTG concentration (0.2 to 1 mM), the biomass and low identities (36.4–81.6%) and small numbers of similar lycopene production both decreased. The highest yield sequences (Table 3). The protein sequences encoded by −1 of 418 mg L lycopene was achieved at 42 h with no these carotenoid biosynthesis genes were also compared IPTG induction. After 42 h of fermentation, the bio- between R12 and other Deinococcus species (Additional mass and lycopene concentration decreased. This file 1: Table S2). The sequence identities were very low decrease may be caused by the consumption of nutri- (27.8–88.5%), and some proteins could not be found in ents, the accumulation of harmful metabolites and the some species (especially lycopene cyclase), which was pressure on strain growth by the highly hydrophobic similar to the result of gene alignment. The carotenoid lycopene stored in the cell membrane (McNerney and biosynthesis genes and proteins of R12 were obviously Styczynski 2017). At the same time, cell lysis and the different from those of other Deinococcus species due instability of lycopene after long-term fermentation can to the low sequence identities and low number of avail- also lead to a decrease of lycopene yield. able strains for alignment. These carotenoid genes and the corresponding proteins from R12 are therefore worth further study. Table 3 Percentages of sequence identity of carotenoid biosynthesis gene sequences between D. wulumuqiensis R12 and other Deinococcus spp. crtE (%) crtB (%) crtI (%) crtLm (%) cruF (%) crtD (%) crtO (%) D. radiodurans R1 85.50 86.30 86.80 82.20 78.70 81.00 90.30 D. gobiensis I-0 78.06 67.70 77.90 72.50 68.70 70.40 80.70 D. actinosclerus BM2 73.20 66.00 74.00 67.40 56.60 71.00 81.60 D. swuensis DY59 75.80 68.90 75.40 62.90 67.00 69.70 81.60 D. soli N5 72.90 67.90 74.90 65.30 63.30 72.10 81.00 D. deserti VCD115 75.10 59.00 75.00 / 38.10 64.60 75.60 D. geothermalis DSM 11300 74.90 65.30 75.00 61.50 61.30 69.40 75.80 D. puniceus DY1 74.90 65.70 73.30 60.00 52.50 64.20 80.50 D. ficus CC-FR2-10 66.30 62.30 74.50 / 36.80 62.30 75.70 D. maricopensis DSM 21211 70.40 62.40 73.10 63.50 59.10 69.30 71.50 D. proteolyticus MRP 61.10 55.80 75.00 / 51.80 63.70 74.50 D. peraridilitoris DSM 19664 51.20 59.20 68.00 / 36.40 62.20 71.80 Xu et al. AMB Expr (2018) 8:94 Page 7 of 13 was increased by the addition of glycerol. Since glyc- 0 mM IPTG erol had already been proved to increase the yield of 0.2 mM IPTG lycopene in previous studies (Kim et al. 2011), dif- 0.4 mM IPTG −1 0.6 mM IPTG ferent concentrations of glycerol (0–100 g L ) were 0.8 mM IPTG added to 2× YT medium (2× YT + G). As shown in 1 mM IPTG −1 Fig. 5, the biomass reached the maximum of 6.45 g L after 30 h, and the lycopene production reached the −1 −1 maximum of 555 mg L after 42 h when 20 g L glycerol was added. However, the content of lycopene gradually decreased when the initial glycerol concen- 2 −1 tration was greater than 20 g L , indicating that the accumulation of lycopene did not require excessive addition of glycerol. Furthermore, cell growth declined rapidly with the increase of initial glycerol concentra- tion, and low levels of biomass limited the lycopene 06 12 18 24 30 36 42 48 54 production. These results demonstrated that among Time (h) the culture media tested in this work, the 2× YT + G −1 medium (20 g L ) was most suitable for the produc- 0 mM IPTG tion of lycopene. 0.2 mM IPTG 0.4 mM IPTG 0.6 mM IPTG The effects of temperature on cell growth and lycopene 0.8 mM IPTG production 1 mM IPTG Temperature is considered the main physical element that directly influences the bacterial growth rate and thus plays an important role in the biosynthesis of carot- enoids. Three temperatures (25, 30, and 37 °C) were assessed according to previous studies (Kim et al. 2009). As shown in Fig. 6, 37 °C was the best temperature for the growth of the EBI strain according to the DCW results. −1 The highest DCW was 7.3 g L at 37 °C after cultivation for 30 h. Moreover, the total lycopene content was much 06 12 18 24 30 36 42 48 54 higher at 37 °C than at 30 or 25 °C. The highest lycopene Time (h) −1 content was 564 mg L at 37 °C after cultivation for 42 h. Fig. 3 Cell dry weight and lycopene yield of strain EBI induced with The DCW and lycopene content were the lowest at 25 °C, different IPTG concentrations. a Cell dry weight of EBI in LB medium and the lycopene yield was also especially markedly lower with the addition of 0–1 mM IPTG at 30 °C for 48 h. b Lycopene production of EBI in LB medium with the addition of 0 to 1 mM IPTG at this temperature. The high biomass obtained at 37 °C at 30 °C for 48 h. The data represent the means of three independent may explain the high lycopene content in the cultures. experiments. Error bars represent standard deviations The lycopene content decreased after 42 h of cultivation, suggesting that cultivation at 37 °C for 42 h is optimal for biomass accumulation and lycopene production. Optimization of the culture medium for lycopene accumulation The effect of light on cell growth and lycopene production To determine the optimal culture medium, LB, 2× YT, Light affects many biological activities such as microbial and SM medium were tested. The lycopene produc- growth, morphogenesis, and biosynthesis of reduced −1 tion of the EBI strain reached 452.49 mg L in 2× YT hydrogen equivalents in living organisms (Chen and −1 medium and 418 mg L in LB medium. By contrast, Chang 2006; Bohne and Linden 2002). In addition, lyco- −1 the yield in SM medium was only 20 mg L (Fig. 4). pene is a light-sensitive product. Therefore, the influ - The effects of additional carbon sources on lycopene ence of light on lycopene biosynthesis was evaluated. The production were investigated by adding different shake flasks were wrapped in silver paper to protect lyco - concentrations of carbon sources to 2× YT medium pene in our system. As shown in Fig. 7, the shake flasks (Table 4). The production of lycopene was inhibited were exposed to 40 W of LEDs to assess the influence of by the addition of starch, lactose, and sucrose, while it lycopene content (mg/L) DCW (g/L) Xu et al. AMB Expr (2018) 8:94 Page 8 of 13 Optimal conditions for lycopene production in shake flasks Based on the results of fermentation optimization, the LB 2× YT temperature was fixed at 37 °C during the entire cultiva - SM tion process, and 2× YT medium was used for seed culti- vation for 12 h. The preculture was then used to inoculate −1 50 mL of fresh 2× YT + G medium (with 20 g L glycerol) in 250-mL shake flasks. Cultivation was conducted in the dark. As shown in Fig. 8, the biomass increased quickly during the first 18 h of cultivation, then increased slowly, −1 and reached a maximum of 7.35 g L at 30 h. The lyco - pene content increased at the beginning, reaching a maxi- −1 mum at 42 h (618 mg L ), and then gradually decreased. Compared with the original conditions, the biomass of EBI increased 1.99 times and the yield of lycopene improved 1.98-fold after optimization. 06 12 18 24 30 36 42 48 54 Time (h) Construction of recombinant plasmids with different crt LB gene order 2× YT The DNA fragments encoding crtE, crtB and crtI were SM 400 amplified and assembled to from the plasmids pET-EIB, pET-BEI, pET-BIE, pET-IBE, and pET-IEB (Fig. 2), which were transferred into E. coli BL21(DE3), resulting in the recombinant strains EIB, BEI, BIE, IBE, and IEB, respec- tively. Acetone extracts from these strains were analyzed for lycopene content by HPLC (Table 5). The strain BEI −1 had the lowest lycopene content of 228 mg L . By con- 100 trast, the lycopene production of the IEB strain reached up −1 to 688 mg L , which was the highest of all six strains and more than three times higher than that of the lowest strain. Discussion 06 12 18 24 30 36 42 48 54 Many efforts have been made to improve the yield Time (h) of lycopene by engineering bacteria, mostly via the Fig. 4 Dry cell weight and lycopene yield of strain EBI in different media. a Cell dry weight from strain EBI in LB medium (black squares), expression of exogenous crtE, crtB and crtI genes for 2× Y T medium (red circles), and SM medium (blue triangles) with lycopene synthesis from Erwinia to Pantoea species. no IPTG, after growth at 30 °C for 48 h. b Lycopene content of strain Yoon et al. constructed engineered E. coli strains har- EBI in LB medium (black squares), 2× Y T + G medium (red circles), boring lycopene genes from Pantoea agglomerans and and SM medium (blue triangles) with no IPTG, after growth at −1 Pantoea ananatis, which produced 60 and 35 mg L 30 °C for 48 h. The data represent the means of three independent experiments. Error bars represent standard deviations of lycopene, respectively (Yoon et al. 2007). When the genes crtE, crtB and crtI from Erwinia uredovora were integrated into Candida utilis, it produced a lycopene light. The strain produced the highest lycopene content −1 yield of 758 μg g DCW (Miura et al. 1998). Mat- −1 (581.2 mg L ) after 42 h of fermentation in the dark, thaus et al. constructed a plasmid harboring crtB and while the biomass was higher under the influence of light. crtI from Pantoea ananatis and transformed Yarrowia −1 The maximum biomass reached 7.23 g L under LED −1 lipolytica, which produced 16 mg g DCW of lyco- lights at 30 h. These results indicate that light has a non- pene (Matthäus et al. 2014). When the lycopene synthe- negligible effect on lycopene accumulation. sis genes from different bacteria were cloned into the Table 4 Eec ff ts of different auxiliary carbon sources (10 g/L in 2× YT medium) on the lycopene yield of the strain EBI Auxiliary carbon source Glucose Glycerol Fructose Starch Lactose Sucrose −1 Lycopene content (mg L ) 371 ± 9.1 481 ± 8.9 449 ± 12.3 295 ± 3.6 183 ± 7.7 214 ± 10.9 DCW 6.2 ± 0.28 5.8 ± 0.12 5 ± 0.3 4.5 ± 0.28 3.3 ± 0.11 4.1 ± 0.17 lycopene content (mg/L) DCW (g/L) Xu et al. AMB Expr (2018) 8:94 Page 9 of 13 lycopene synthesis genes from extremophilic radia- 0 g/L glycerol tion-resistant microorganisms were rarely investigated. 10 g/L glycerol a In this work, the lycopene synthesis genes from the 20 g/L glycerol 40 g/L glycerol recently isolated extremophilic microorganism Deino- 60 g/L glycerol coccus wulumuqiensis R12 were analyzed and cloned in 80 g/L glycerol 100 g/L glycerol E. coli. The transgenic E. coli strain EBI produced a high content of lycopene after twin optimization of fermen- tation conditions and gene expressing levels (Fig. 9), and thus provides a new microbial gene source for lyco- pene synthesis and lays a good foundation for improv- ing lycopene production in engineered Escherichia coli. In prokaryotic expression systems, the strong inducer IPTG exacerbates the toxicity of haloalkane substrates, causing damage to the E. coli host, which often bears a metabolic burden due to the recombinant plasmid it con- tains. Excess IPTG can result in non-trivial economic 06 12 18 24 30 36 42 48 54 losses and toxic effects, including reduced cell growth Time (h) and lower recombinant protein concentration (Papaneo- b phytou and Kontopidis 2014). In our study, when IPTG 0 g/L glycerol 10 g/L glycerol was not added at all, the lycopene content and cell growth 20 g/L glycerol were close to the highest. With the increase of IPTG con- 40 g/L glycerol centration, the lycopene content and cell growth gradu- 60 g/L glycerol 80 g/L glycerol ally decreased. Under high levels of protein production, 100 g/L glycerol the E. coli cells bear a negative pressure known as the metabolic burden or metabolic load, which is attributed to the overconsumption of metabolic precursors (e.g., amino acids, adenosine triphosphate, FPP) to form non- essential foreign proteins, as well as the maintenance and replication of recombinant plasmid vectors (Dvorak et al. 2015; Mairhofer et al. 2013). Low IPTG concentrations can result in efficient induction, and leaky expression sometimes occurs even when IPTG is not added, which 06 12 18 24 30 36 42 48 54 allows for sufficient expression of genes within the path - Time (h) way to achieve a good yield. Similar inducer concentra- tions that allow full gene expression have been reported Fig. 5 Dry cell weight and lycopene yield of strain EBI with different initial concentrations of glycerol. a Cell dry weight of EBI in 2× Y T (Kim et al. 2011; Bahieldin et al. 2014; Kim et al. 2009; −1 medium with the addition of 0–100 g L of glycerol after growth at Zhang et al. 2015b). In some cases, tuning the IPTG con- 30 °C for 48 h. b Lycopene production of EBI in 2× Y T medium with centration by reducing it dramatically or even not adding −1 the addition of 0–100 g L of glycerol after growth at 30 °C for 48 h. any inducer can improve the host’s fitness, although the The data represent the means of three independent experiments. mechanism driving the induction of T7 RNAP expression Error bars represent standard deviations in the absence of IPTG is not clear. Here, we showed that culturing E. coli cells in LB medium in the absence of the inducer IPTG could provide a cost-effective, simple and pGAPZB plasmid and introduced into Pichia pastoris competitive alternative for the production of lycopene. X33, the recombinant strain showed a lycopene pro- Optimization of the culture medium is a useful method −1 duction of 73.9 mg L (Bhataya et al. 2009). Bahieldin to enhance lycopene production. In this study, the use of et al. constructed a plasmid harboring the crt genes glycerol as an auxiliary carbon source greatly improved from Pantoea ananatis under the control of the ADH2 lycopene production, which may be due to a higher ace- promoter and introduced it into Saccharomyces cer- tate concentration in the cultures grown on glucose than −1 evisiae, which produced a yield of 3.3 mg lycopene g in the ones grown on glycerol. At high concentrations, DCW (Bahieldin et al. 2014). Thus, diverse sources of acetate acts as an inhibitory metabolite, lowering carote- lycopene synthesis genes expressed in different hosts noid production. Moreover, glucose has been reported to resulted in different lycopene yields. However, the catabolically repress the T7 promoter in the recombinant lycopene content (mg/L) DCW (g/L) Xu et al. AMB Expr (2018) 8:94 Page 10 of 13 with light 8 25 °C without light 30 °C 8 DCW with light DCW without light 37 °C 06 12 18 24 30 36 42 48 54 Time (h) Fig. 7 Dry cell weight and lycopene yield of strain EBI with or 06 12 18 24 30 36 42 48 54 without light. The shake flasks were exposed to LEDs (filled squares), Time (h) or were incubated and without light (open circles) at 37 °C for 48 h 25 °C 30 °C 37 °C DCW lycopene content 06 12 18 24 30 36 42 48 54 -100 06 12 18 24 30 36 42 48 54 Time (h) Time (h) Fig. 6 Dry cell weight and lycopene yield of strain EBI at different Fig. 8 Dry cell weight and lycopene yield of strain EBI under −1 temperatures. a Cell dry weight of EBI in 2× Y T + G medium (20 g L optimized conditions. Dry cell weight (filled squares) and lycopene glycerol) after growth at 25, 30, and 37 °C for 48 h. b Lycopene content (open squares) of the EBI strain under the optimal conditions −1 production of EBI in 2× Y T + G medium (20 g L glycerol) after in shake flask culture. The values are the averages ± standard growth at 25, 30, and 37 °C for 48 h. The data represent the means deviations from three independent experiments of three independent experiments. Error bars represent standard deviations slow down. Conversely, appropriately high temperatures system we used for lycopene synthesis (Yang and Guo can promote cell growth, balance enzyme expression 2014; Guzman et al. 1995). and increase the activities of enzymes. It is well-known Temperature is one of the most important environ- that carotenoids are important for the protection against mental factors affecting the growth and development of photo-oxidative damage in non-photosynthetic organ- E. coli. In protein expression systems based on E. coli, isms. Many non-phototrophic bacteria and fungi rely on temperature affects both induction and protein expres - carotenoids for protection when growing exposed to light sion. Although it was found that lower temperatures and air (Marova et al. 2012). As with other carotenoids, favor more lycopene formation (Kim et al. 2009; Vadali the stability of lycopene is affected by light. Under illu - et al. 2005; Lee et al. 2004), when the strain EBI was mination, lycopene decomposes via isomerization and grown at 37 °C the lycopene content and DCW were oxidation, which protects the cells from oxidative dam- both higher than at either 30 or 25 °C. Low tempera- age caused by exposure to strong light, but also decreases tures decrease the rate of nutrient consumption, and the concentration of lycopene in the cells (Hernández- thus some metabolic processes, such as protein synthesis, Almanza et al. 2016). lycopene content (mg/L) DCW (g/L) DCW (g/L) lycopene content (mg/L) lycopene content (mg/L) DCW (g/L) Xu et al. AMB Expr (2018) 8:94 Page 11 of 13 Table 5 Lycopene production of the six recombinant strains with different crt gene order Strain EBI EIB BEI BIE IEB IBE −1 Lycopene content (mg L ) 605 ± 12 583 ± 15 228 ± 9 373 ± 16 688 ± 10 529 ± 18 The strains are named according to the gene order of E: crtE, B: crtB, and I: crtI expression requires the orderly arrangement of genes to balance their translation levels. Combined with the size and expression of enzymes, a high level of synergy is needed to achieve higher yields. Phytoene desaturase (PDS, encoded by crtI), the first enzyme involved in phytoene conversion to colored carotenoids, catalyzes a rate-limiting step in carotenoid biosynthesis (Chamovitz et al. 1993). The catalytic func tions of bacterial phytoene desaturases are diverse, which can lead to low lycopene concentrations because of its poor catalytic specificity. Stickforth et al. demonstrated that high phytoene desaturase concentrations or a low phytoene supply favor the formation of lycopene (Stick forth and Sandmann 2007). Ostrov et al. introduced the Fig. 9 Lycopene production was improved by the combined lycopene production pathway into a modular biosensor optimization of culture conditions and gene order. The E. coli strain and found that after adding two copies of lycopene syn- EBI produced a high content of lycopene after twin optimization of thase (encoding by crtI), lycopene production increased fermentation conditions and gene expressing levels more than three times (Ostrov et al. 2017). Among the six strains in our study, the lycopene yield of IEB was the highest. This is probably due to the fact that the crtI gene The efficiency of multi-gene expression systems is was closest to the promoter, which increased its transla mainly affected by promoters, transcription factors, and tion efficiency and the final substrate conversion rate to translation levels. Nevertheless, the gene order is also lycopene. At the same time, lycopene is only synthesized important. Within an operon, the transcription efficiency from FPP after successful multi-gene expression of crtE, of a gene decreases as its position moves away from the crtB and crtI, which means that balanced gene expression promoter. The expression of a gene at the first position is needed to avoid excessive accumulation of intermedi is therefore higher than that of an identical gene at the ate products that can inhibit cell growth. second position, which should be higher than that of an In conclusion, a recombinant strain with a new source identical gene at the third position, and so on (Han et al. of lycopene synthesis genes from the radiation resistant 2011). A novel approach for metabolic pathway optimi- microorganism Deinococcus wulumuqiensis R12 was con- zation, oligo-linker mediated assembly (OLMA), was structed. We found some important differences between applied in the lycopene synthetic pathway to swap the these lycopene synthesis genes and other homologous order of crtE, crtB and crtI, which led to selection of the microbial genes, which merits further study. After opti- best strain EBI, the lycopene yield of which was 36 times mization of culture media, temperature and illumination, higher than that of the least productive strain IEB (Zhang −1 the lycopene content of strain EBI reached 618 mg L in et al. 2015a). In our study, the productivity of strain IEB −1 2× YT + G medium (with 20 g L glycerol), after 42 h of was 3 times higher than that of the least productive strain fermentation in the dark at 37 °C. Finally, six recombinant BEI, which suggested that the order of genes had a great strains with different crt gene orders were constructed, influence on lycopene synthesis. An improper gene order −1 and the highest lycopene content was 688 mg L in can result in a severe imbalance in the pathway, which in strain IEB, which was about three times higher than that turn affects the product yield. Through sequential con - of the lowest strain BEI, underscoring the effect of gene trol of the downstream, upstream, and competing path- regulation on lycopene synthesis. Taken together, the ways of farnesyl diphosphate (FPP) via a predetermined strain IEB was improved 2.2-fold compared to the origi order of key genes in the crucial metabolic node in the nal recombinant strain EBI. Our results will provide new biosynthesis of terpenoids, a carotenoid production of −1 −1 guidance for the synthesis, regulation and industrial pro- 1156 mg L (20.79 mg g DCW) was achieved (Xie duction of lycopene and other carotenoids. et al. 2015). These strategies indicate that multi-gene Xu et al. AMB Expr (2018) 8:94 Page 12 of 13 References Additional file Alper H, Miyaoku K, Stephanopoulos G (2005) Construction of lycopene- overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23:612–616 Additional file 1: Figure S1. 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