Background

Astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione), a red-color carotenoid, is one of the strongest antioxidants in nature [1], exhibiting tremendous potential for applications in healthcare and pharmaceuticals due to its antioxidant [2], anti-inflammatory [3] and anti-cancer activity [4]. It has been widely used in aquaculture, food, cosmetic and pharmaceutical industries [5]. According to the analysis of Stratistics Market Research Consulting (MRC), the global astaxanthin market is accounted for $695.94 million in 2020 and is expected to reach $1356.47 million by 2028 growing at a compound annual growth rate of 8.7% [6]. Currently, commercial astaxanthin production relies on two routes, chemical synthesis and extraction from nature producers (such as Haematococcus pluvialis algae) [6]. However, the former route of production is not sustainable and poses concerns in health and food safety, while the latter is very costly (> $7000 per kg) [7].

To lower the cost, many studies focused on the production of astaxanthin from an inexpensive carbon source using microbes. Escherichia coli [8,9,10,11,12,13,14,15,16,17], Saccharomyces cerevisiae [18,19,20,Library screening

Because astaxanthin but not the other carotenoids can efficiently diffuse through the cell membranes [41], a colorimetry of culture media (measured at 515 nm) was developed to be used for the screening of high astaxanthin-producing strains [15]. Cells from the library were diluted and then spread on LB plates. Single colonies with red color were inoculated in individual wells of a 48 deep-well microplate (4.6 mL) containing 1 mL of LB medium and incubated at 30 °C and 1000 rpm for 48 h on an MBR-420FL shaker (TAITEC, Japan). Two hundred microliter of the bacterial culture were centrifugated at 5000×g for 10 min. Then, the supernatant was transferred into a 96-well plate in which the OD515 was read using a SynergyNeo2 multi-mode reader (SynergyNeo2, BioTek, USA).

Whole-genome resequencing and data analysis

Total genomic DNA of E. coli E. coli AST-4AS was extracted from mid-log phase bacterial cultures according to the manufacturer’s protocol using the TIANamp Bacterial DNA Kit (Tiangen Biotech Co., Bei**g, China). Genomic library construction and whole-genome resequencing were performed on the Illumina NOVAseq platform by Sangon Biotech (Shanghai, China). The paired-end reads from E. coli E. coli AST-4AS were aligned to the reference genome of E. coli MG1655 using BWA software (Burrows-Wheeler Aligner, version 0.7.17). Mutations, including base substitutions, deletions, and insertions, were detected by SAMtools (version 1.9), MarkDuplicates (version 4.1.1.0), and BEDTools (version 2.28.0). DNA frameshift mutations were further validated by PCR and sequencing.

CRISPRa and CRISPRi of the mutant genes

Mutant genes with CRISPR activation and interference were performed as described by Niu et al. [38]. The pTargetA-X series used in targeted single-gene activation or repression, with N20 sequences targeting gene loci of interest, was obtained by inverse PCR from pTargetA using primers targeting N20F/N20 genes, then cut with KpnI and self-ligated. For activation, the N20 sequence was designed to target the non-template strand upstream of the promoter. For repression, the N20 sequence was designed to target the 5′ end (about 100 bp downstream of ATG) of the gene on the non-template DNA strand. As the scRNA fragment was flanked by BamHI and BglII, the scRNA expressing vector pTargetA-XY could be reassembled from pTargetA-X and pTargetA-Y using the standard BglBrick assembly approach.

To investigate the effects of activation and repression on growth and astaxanthin production, pBbB2K-dCas9*-MCPSoxS and pTargetA-X were co-transferred into E. coli AST-4. A single colony was grown in 5 mL LB (with 1% glucose) in a falcon tube at 37 °C overnights. The overnight cultures were inoculated into 10 mL of the fermentation medium at 37 °C until the OD600 reached 0.8, anhydrous tetracycline was added to the media at a final concentration of 200 nM to induce the expression of dcas9*, and then further cultured for 72 h.

Astaxanthin production

A single colony was inoculated into 5 mL of LB medium in a falcon tube, which was incubated overnight at 30 °C. The overnight seed culture was then inoculated into 50 mL of SBMSN medium with an initial OD600 of 0.1. SBMSN medium (pH 7.0) contained (per liter) 12 g peptone, 24 g yeast extract, 1.7 g KH2PO4, 11.42 g K2HPO4, 1 g MgCl2⋅6H2O, 1.42 g ammonium oxalate, and 2 g Tween-80. The cultures were incubated at 30 °C for 48 h in a rotary shaking incubator set to 150 rpm. Cell growth was measured according to the OD600 and converted into DCW (g/L) using a standard curve.

Fed-batch fermentation was performed in a 2 L fermenter (MiniBox 2 L*2 Parallel Bioreactor System, T&J Bioengineering (Shanghai) Co. LTD, Shanghai, China) containing 1.2 L of SBMSN medium with an initial OD600 of approximately 0.1. The temperature was controlled at 30 °C, and the pH value was maintained at 7.0 by the automatic addition of NH4OH. The airflow rate was 2 L/min. Dissolved oxygen was kept above 25% by adjusting the agitation rate from 400 to 1200 rpm. A feed solution (pH 7.0) containing (per liter) 500 g glucose, 15 g peptone, 30 g yeast extract and 30 g MgSO4·7H2O was fed continuously to the fermenter using a pH–stat feeding strategy. Once the glucose was depleted, the pH rose rapidly. When the pH was greater than 7.1, the feed was automatically added to the fermenter. Samples were periodically withdrawn, and these parameters (OD600 and astaxanthin concentrations) were determined.

Quantitative real-time PCR (qRT-PCR)

The total RNA from E. coli cells grown in the fermentation medium for 36 h in shake flasks was isolated using an RNA extraction kit (Dongsheng Biotech, Guangzhou, China) following the manufacturer’s instructions. The first-strand cDNA was synthesized using an All-in-One™ First-Strand cDNA Synthesis kit (GeneCopoeia, Guangzhou, China). Quantitative real-time PCR was performed with the All-in-One™ qPCR Mix kit (GeneCopoeia) by an iCycler iQ5 Real Time PCR system (Bio-Rad Laboratories, California, USA). One hundred ng of cDNA was added as a template. The PCR program was set as follows: 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 15 s. The expression levels were analyzed by the 2−ΔΔCt method described by Livak et al. [42] and normalized by cysG gene expression as reference. Three biological replicates for each sample were used for qRT-PCR analysis, and three technical replicates were analyzed for each biological replicate.

Replacement of gene

Gene knockout or replacement was performed according to the CRISPR-Cas method as previously described [17, 34, 43, 44]. The sgRNA plasmid pTargetB-yadC for replacement was obtained as described above for the CRISPRi system. The rnb was amplified from E. coli by PCR and then inserted into KpnI/ApaI-digested pZSKBP to obtain pZSKBP-rnb. The upstream and downstream homology arms of the yadC were amplified and then successively inserted into the MluI/AvrII and ApaI/SmaI of pZSKBP-rnb. The targeting fragment was cut from the above plasmid using MluI/SmaI and then transferred into the electrocompetent cells harboring pCas* and pTargetB-yadC to replace the corresponding gene.

Extraction and quantification of carotenoids

Cells were extracted with acetone to isolate carotenoids as previously described [9, 17]. E. coli cultures (250 μL) were harvested by centrifugation at 13,523×g for 5 min. The cell pellet was washed with water and extracted with 1 mL of acetone at 55 °C for 15 min with intermittent vortexing. The acetone supernatant after centrifugation was transferred to a new tube. Carotenoids were analyzed by HPLC (Shimadzu HPLC system, Model LC-20A, Shimadzu, Japan) using an Inertsil ODS-SP column (5 μm, 4.6 × 150 mm, GL Sciences Inc., Tokyo, Japan). The mobile phase was acetonitrile–methanol (65:35 v/v) at a flow rate of 1 mL/min. The absorbance of carotenoids at 477 nm was detected using a photodiode array detector (SPD-M20A). Carotenoid compounds were identified based on their retention times relative to standard compounds (Sigma-Aldrich, St. Louis, MO, USA). Astaxanthin was quantified by comparing the integrated peak areas with those of authentic standards.

Statistical analysis

All experiments were conducted in triplicate, and data were averaged and presented as the means ± standard deviation. Oneway analysis of variance followed by Tukey’s test was used to determine significant differences using the OriginPro (version 9.0) package. Statistical significance was defined as p < 0.05.