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High-yield porphyrin production through metabolic engineering and biocatalysis

Nature Biotechnology (2024)Cite this article

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An Author Correction to this article was published on 02 July 2024

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Abstract

Porphyrins and their derivatives find extensive applications in medicine, food, energy and materials. In this study, we produced porphyrin compounds by combining Rhodobacter sphaeroides as an efficient cell factory with enzymatic catalysis. Genome-wide CRISPRi-based screening in R. sphaeroides identifies hemN as a target for improved coproporphyrin III (CPIII) production, and exploiting phosphorylation of PrrA further improves the production of bioactive CPIII to 16.5 g L−1 by fed-batch fermentation. Subsequent screening and engineering high-activity metal chelatases and coproheme decarboxylase results in the synthesis of various metalloporphyrins, including heme and the anti-tumor agent zincphyrin. After pilot-scale fermentation (200 L) and setting up the purification process for CPIII (purity >95%), we scaled up the production of heme and zincphyrin through enzymatic catalysis in a 5-L bioreactor, with CPIII achieving respective enzyme conversion rates of 63% and 98% and yielding 10.8 g L−1 and 21.3 g L−1, respectively. Our strategy offers a solution for high-yield bioproduction of heme and other valuable porphyrins with substantial industrial and medical applications.

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Fig. 1: Identification and quantitatively analysis of CPIII and Fe-CPIII in different R. sphaeroides strains with fnrL gene deletion.
Fig. 2: Engineering of R. sphaeroides HY01 for enhanced CPIII production through CRISPR–dCas12a-derived genome-wide screening.
Fig. 3: Fine-tune timing of PrrA phosphorylation boosts CPIII production in a 5-L bioreactor.
Fig. 4: In vitro biosynthesis of metalloporphyrins by enzymatic catalysis.
Fig. 5: Screening and engineering HemQ for efficient conversion of CPIII to heme.
Fig. 6: Bioproduction of heme and Zn-CPIII in a 5-L bioreactor.

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Data availability

The authors declare that the primary data supporting the findings of this study are accessible within the paper and its Supplementary Information files. Additional data can be obtained from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The developed code in this work is available at https://codeocean.com/capsule/8302979/tree/v1 or https://doi.org/10.24433/CO.8752603.v1.

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Acknowledgements

We thank Q. Chen (School of Biotechnology, East China University of Science and Technology), L. Zhang (School of Chemistry and Molecular Engineering, East China Normal University) and W. Zhang (State Key Laboratory of Microbial Technology, Shandong University) for their technical support in enzyme mining, engineering and characterization. We are also deeply grateful to Z. Zhou (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) for her generous gift of the ZX-5 strain and to Inner Mongolia Kingdomway Pharmaceutical Co., Ltd. for providing the HY01 strain. We would like to extend our gratitude to D.-Q. Lin from the College of Chemical and Biological Engineering at Zhejiang University for his invaluable guidance in conducting the techno-economic analysis and for providing assistance in using the SuperPro Designer simulation software. During the manuscript preparation, we also greatly appreciate the constructive insights provided by J.-J. Zhong, L. Bai and T. Liu from the School of Life Sciences and Biotechnology, Shanghai Jiao Tong University. This work was financially supported, in part, by grants from the National Key Research and Development Program of China (2020YFA0907800 to Z.L. and 2020YFA0907304 to T.G.-Y.); the National Natural Science Foundation of China (32121005 to Z.L. and 32370064 and 31870040 to T.G.-Y.); 2023 Double World-Class Project-Key Program-Intelligent Biomanufacturing to Z.L.; and the Open Funding Project of the State Key Laboratory of Microbial Metabolism (MMLKF22-03 to T.G.-Y.).

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  1. These authors contributed equally: Haihong Chen, Yaohong Wang, Weishan Wang, Ting Cao, Lu Zhang.

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering and School of Biotechnology, East China University of Science and Technology, Shanghai, China

    Haihong Chen, Yaohong Wang, Ting Cao, Lu Zhang, Zhengduo Wang, Xuran Chi, Tong Shi, Huangwei Wang, Xinwei He, Mindong Liang, Mengxue Yang, Wenyi Jiang, Dongyuan Lv, Jiaming Yu, Guoliang Zhu, Yongtao Xie, Bei Gao, Xinye Wang, Xueting Liu, Youyuan Li, Liming Ouyang, Jingyu Zhang, Huimin Liu, Gao-Yi Tan & Lixin Zhang

  2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Weishan Wang & Zilong Li

  3. State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Yaojun Tong

  4. Key Biosensor Laboratory of Shandong Province, Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China

    Xuekui Xia

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Contributions

G.-Y.T. and Lixin Zhang supervised the project. G.-Y.T., Lixin Zhang and W.W. designed the experiments. Lu Zhang, T.S., X.H., M.Y., W.J. and D.L. engineered and optimized R. sphaeroides. Z.W. and H.C. designed the crRNA library and screened the strains. T.C., X.C., H.C., Z.W., X.W. and B.G. mined the enzyme and performed enzymatic catalysis. Y.W., X.C. and M.L. developed the heme detection method and conducted high-throughput screening work. H.W., H.C., G.Z., X.L., J.Z., H.L., X.X. and Y.X. isolated, prepared and identified chemical compounds. H.C., Z.W., L.O., Y.L., J.Y. and Z.L. conducted the fed-batch work in bioreactor. H.C., Z.W. and Z.L. developed the pilot-scale fermentation process and the method for isolating and purifying CPIII. Z.W. and H.C. conducted the techno-economic analysis. G.-Y.T. wrote the manuscript. Lixin Zhang, G.-Y.T. and Y.T. edited the manuscript.

Corresponding authors

Correspondence to Gao-Yi Tan or Lixin Zhang.

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Competing interests

G.-Y.T, Lixin Zhang, W.W., Lu Zhang, Y.W., T.C. and H.C. filed eight Chinese patent applications (201910999107.4, 202211452809.9, 202211487904.2, 202310737429.8, 202310738915.1, 202310750263.3, 202310754364.8 and 2023118130742) based on this work. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Porphyrin pathway-derived products or intermediates and the verification of CPIII production in fermentation broth.

a, The chemical structures of key products or intermediates in porphyrin biosynthetic pathway. 5-ALA, 5-Aminolevulinate; UPENIII, Uroporphyrinogen III; CPENIII, Coproporphyrinogen III; CPIII, Coproporphyrin III; Fe-CPIII, Fe-coproporphyrin III; VB12, Vitamin B12; PPIX, Protoporphyrin IX; Bchl, Bacteriochlorophyll. b, Structure verification of CPIII in fermentation broth sample. i) HPLC profile of sample and standard CPIII; ii) MS/MS spectra with 50-eV collision energy for purified and standard CPIII.

Extended Data Fig. 2 Construction and PCR verification of various mutants and stepwise enhancement of CPIII production in R. sphaeroides.

a, Construction and PCR verification 2.4.1-ΔfnrL. b, Construction and PCR verification of ZX-5-ΔfnrL. F1 and F2 are the PCR products that were amplified using ZX-5-ΔfnrL-up-ck-F/R and ZX-5-ΔfnrL-dn-ck-F/R, respectively. c. Construction and PCR verification of HY01-ΔfnrL. d. Construction and PCR verification of HY01-ΔhemN. F3 and F4 are the PCR products that were amplified using HY01-ΔhemN-up-ck-F/R and HY01-ΔhemN-dn-ck-F/R. e. Construction and PCR verification of HY01-ΔhemN-prrABpLB2. F5 and F6 are the PCR products that were amplified using ΔhemN-prrAB-up-ck-F/R and ΔhemN-prrAB-dn-ck-F/R. The PCR verifications in b-c were performed three times each (n = 3; replicates are shown in Source Data files; in each replicate, at least two double crossover conjugants were selected). f. Heat map depicts the differentially expressed PrrA target operons (selected) during fermentation in bioreactor at 16, 24, 40 and 60 h. This result indicated that three genes associated with the porphyrin metabolism pathway, namely hemA, hemC, and hemE, which are regulated by prrA, exhibited a substantial upregulation in transcription levels at the 40 h of fermentation compared to the 24 h. FPKM (fragments per kilobase of transcript per million fragments mapped) values were taken as generation of the heat map by origin 2022 software. g. Stepwise enhancement of CPIII production in R. sphaeroides (n = biologically independent replicates). *: Fed-batch fermentation in 5-L bioreactor; #: IPTG addition. Bar graph with error bars represents mean ± s. d.

Source data

Extended Data Fig. 3 Verification of metalloporphyrin production by HRMS and biocatalytic synthesis of Zincphyrin.

a-d, Identification of Co-CPIII, Ni-CPIII, Cu-CPIII, and Mn-CPIII by high-resolution mass spectrometry (HRMS). All the accurate mass measurements for impurities were within < 5 ppm of the theoretical m/z value. e, HPLC profile of the enzymatic catalysis of CPIII for the in vitro synthesis of Zincphyrin using HemHSa and zinc sulfate. The reaction is conducted in Tris-HCl buffer (pH 8.0) at 37 °C under the following conditions: i) In the reaction system, only 50 μM CPIII is present. ii) In condition i, 50 μM ZnSO4 and 1 μM HemHSa were added.

Extended Data Fig. 4 Methodological characterization and analysis of CRISPR-Cas12a-based heme detection.

a. Fluorescence intensity of detection system in the presence of heme and its structural analogues (n = 3 biological independent detection of fluorescence intensity per condition). Fluorescence intensity was measured in the presence of heme, coproporphyrin I, coproporphyrin III, Fe-coproheme III, and protoporphyrin, with a concentration of 200 nM for heme and 2000 nM for each of the remaining porphyrin compounds. b. The linear relationship between the concentration of heme and the cleavage rate. The concentration range in which the calibration curve was linear was 10–200 nM (n = 3 biological independent replicates; R2 > 0.993). Bar graphs with error bars in a and b represent mean ± s. d. c. Analysis of the reproducibility and accuracy of heme detection method. Three different concentrations of heme samples (sample 1, 2, 3) were independently tested in six times (n = 6) using a 96-well plate. The coefficient of variation (c.v.) and sample recovery (Rec.) rate were calculated for the detection results.

Source data

Extended Data Fig. 5 Mining HemQ homologs and comparing crude enzyme activities.

a. Mining of hemQ homologous (NCBI blast 2.11.0+) by using maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. The tree with the highest log likelihood (−1234833.12) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 1000 amino acid sequences. There were a total of 1761 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (version: 10.2.6). Utilizing hemQ from Bacillus subtilis (P39645) as a probe sequence, a total of 30 candidate genes were selected. The codon optimization and complete gene synthesis for the selected candidates were conducted by GenScript Inc (Nanjing, China). E. coli was employed for recombinant protein expression and subsequent activity assessment. b. The crude enzyme activity of HemQ28 compared to different previously reported HemQ homologous proteins. HemQBs, HemQCg, HemQSa, and HemQMt are derived from Bacillus subtilis, Corynebacterium glutamicum, Staphylococcus aureus, and Mycobacterium tuberculosis, respectively. All these proteins were recombinantly expressed in E. coli and prepared as corresponding crude enzyme solutions. The enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the conversion of 1 μM substrate or generate 1 μM product within one minute under optimal temperature (37 °C) and pH conditions. This study used bovine serum albumin (BSA) at a known concentration as a reference and obtained bands of the target protein through SDS-PAGE. Subsequently, the concentration of the target protein was estimated by comparing its band intensities with those of BSA using BioRad Image Lab software (version: 6.0.0 build 25). Three biologically independent replicates (n = 3) were conducted for enzymatic activity detection of each HemQ homologue. Bar graph with error bars represent mean ± s. d. *P < 0.05.

Source data

Extended Data Fig. 6 Error-prone mutagenesis and screening of HemQ28 variant.

The process of mutation and screening includes: error-prone PCR, DNA assembly, plasmid transformation, cell culture and preservation, protein expression, enzymatic reaction and heme extraction, biosensing of heme, and detection and analysis.

Extended Data Fig. 7 Biocatalysis of heme by CPIII and the stability assessment of HemQ28-M.

a, b, Quantitative analysis of heme and other chemicals after enzymatic catalysis (n = 3 biologically independent replicates). c, d, Effects of temperature and pH on the enzymatic activity of HemQSa and HemQ28-M. Three biologically independent replicates (n = 3) were conducted for each condition in c and d. e, One-time addition of crude enzyme catalyzed the conversion CPIII to heme in 5-L bioreactor. At the beginning of the reaction, 2 L of CPIII with a concentration of 18 g/L was added all at once. The crude enzyme supernatant obtained by crushing 180 g of wet recombinant E. coli cells was added to the reaction system at the beginning of the reaction (that is, 0 h). The reaction was performed at 37 °C and pH 7.0 with agitation at 100 rpm. f, Heme production rate after fed-batch addition of crude enzyme solution (CES) in 5-L bioreactor. After the enzymatic reaction commenced, the CES obtained by crushing wet recombinant E. coli cells was fed-batch added to the 5-L bioreactor at 1, 3, 5, and 7 h. This study measured the heme production rate within two hours after the addition of CES at 1, 3, and 5 h. The concentration of the target fusion protein in the CES was determined by comparing its band’s grayscale intensity with that of a BSA standard following SDS-PAGE electrophoresis, using a concentration of 0.5 g/L BSA as a reference for quantification. Each addition of CES contains 722 ± 140 mg of target fusion protein (derived from approximately 36 g of wet E. coli cells). After each addition of CES, heme concentration increases were measured at five intervals (specifically, 0–5 min, 5–10 min, 25–30 min, 55–60 min, and 115–120 min) within the subsequent two hours to calculate the heme production rate for each corresponding time interval. Bar graphs with error bars represent mean ± s. d. *P < 0.05.

Source data

Extended Data Fig. 8 Purification and preparation of CPIII from fermentation broth through crystallization.

a, Proposed workflow for the purification and preparation of CPIII through crystallization. b, Effects of precipitation pretreatment on crystallization efficiency (three independent replicates were conducted under each condition). Control: direct crystallization using fermentation broth supernatant; NaOH: crystallization after alkaline precipitation (pH 8.5) using NaOH; IPA: addition of 20% isopropanol to the fermentation broth supernatant for alcohol precipitation followed by crystallization; NaOH-IPA: crystallization after combined alkaline and isopropanol precipitation. c, Effects of precipitation pretreatment and acidic acetone extraction on crystallization efficiency (three independent replicates were conducted under each condition). After combined alkaline precipitation and isopropanol precipitation (NaOH-IPA), the obtained supernatant undergoes crystallization following treatment with 30% acidic acetone. CPIII purity and recovery rates were calculated separately according to the Method section. d–g, Effect of pH, CPIII concentration, temperature, and crystallization time on the purity or recovery rate of CPIII. The CPIII fermentation broth supernatant was applied to optimized crystallization conditions. Purity and recovery rates were calculated separately following the procedures outlined in the Method section. h, Effects of recrystallisation time on the purity or recovery rate of CPIII. i, Effect of primary crystallization and recrystallization on the purity and recovery rate of CPIII (three independent replicates were conducted under each condition). CPIII purity and recovery rates were calculated separately according to the Method section. j, The HPLC quantitative standard curve for CPIII. CPIII standard (#C882263; Macklin3 Inc, Shanghai, China) of 1 mg was dissolved in 1 mL of DMSO to prepare a stock solution with a concentration of 1000 mg/L. Under subdued light conditions, the CPIII stock solution was further diluted in a series of concentrations using DMSO, resulting in solutions with concentrations of 125 mg/L, 62.5 mg/L, 31.25 mg/L, 15.625 mg/L, and 7.8125 mg/L. Each concentration was prepared in triplicate. The peak areas of CPIII at 390 nm were determined using HPLC. The peak area values were obtained for each concentration, and the data represent mean ± s. d. of three replication. k, The HPLC profiles of purified CPIII sample and the CPIII standard. Based on the CPIII standard curve calculation, the purity of CPIII samples was determined to be 95.2%.

Source data

Extended Data Fig. 9 Simulation of large-scale CPIIII and heme bioproduction processes for techno-economic analysis.

This model is designed to simulate the large-scale production of CPIII crystals (with a CPIII content of at least 95%, w/w) and heme crystals (with a heme content of at least 93%, w/w). The process for CPIII production consists of two sections: CPIII fermentation for obtaining CPIII fermentation broth, and CPIII preparation for obtaining CPIII crystal product via purification and crystallization. The annual production rate is 20 tons of CPIII. According to the model, the installation is planned to operate 24 hours a day, for a total of 329 days per year (the remaining days were allocated for maintenance and conservation tasks). The annual batch throughput for producing heme is 69 batches, with each batch time and cycle time is 127 and 114 h, respectively. The process for heme production comprises five sections: CPIII fermentation to obtain CPIII fermentation broth (shared with CPIII production), CPIII preparation to obtain CPIII crystal products through purification and crystallization (shared with CPIII production), E. coli culture to cultivate E. coli cells expressing recombinant fusion proteins in fermenters, CES preparation involves cell storage and disruption to obtain cell lysate supernatant containing recombinant fusion proteins, and the Heme preparation section to catalyze the conversion of CPIII into heme using CES and subsequently purify and crystallize to obtain the final heme product (crystallization and purification equipment and lines are shared with CPIII production). The annual production rate is 16 tons of heme. According to the model, the installation is planned to operate 24 hours a day, for a total of 330 days per year (the remaining days were allocated for maintenance and conservation tasks). The annual batch throughput for producing heme is 69 batches, with each batch time and cycle time is 148 and 114 h, respectively. The modeling process utilizes parameters or data from both the built-in database of SuperPro® Designer simulation software and project-specific data from both the experimental results of this study and the Supplementary Table. 4.

Extended Data Fig. 10 Techno-economic analysis of large-scale CPIII and heme bioproduction.

a, Breakdown of annual operating cost for CPIII and heme production. i, Annual cost breakdown by section (%) for CPIII production. ii, Annual operating cost breakdown (%) for CPIII production. iii, Annual cost breakdown by section (%) for heme production. iv, Annual operating cost breakdown (%) for heme production. In this model, the total annual operating costs for CPIII and heme are $935,000 and $2,512,000, respectively (Supplementary Table. 5). The data in panels i and iii are derived from Itemized Cost analyses conducted within this model, while the data in panels ii and iv stem from the Economic Evaluation analysis. The facility-dependent cost encompasses expenses for maintenance, depreciation, and miscellaneous costs. Labor-Dependent Cost: comprises all labor-dependent operating expenses excluding those for laboratory analyses, quality control, and quality analyses. Laboratory/QC/QA Cost: covers laboratory analyses, quality control, and quality analyses. Consumables Cost: certain equipment necessitates the use of consumables; this cost category includes expenses for periodically replaced materials. Utilities: Heating Agents, Cooling Agents, and Power Types. The modeling process utilizes parameters or data from both the built-in database of SuperPro® Designer simulation software and project-specific data from both the experimental results of this study and the Supplementary Table. 4. b. The annual average price and gross margin of heme in the international trading market over the last six years (2018–2023). The information related to the price and gross margin of heme in the international trading market is based on the 2023 Heme Market Research Report, a commercially available market research report provided by MARKET MONITOR Co., Ltd, Changsha, China. The Market Research Report was published on July 15, 2023, with the report code GMM2292280. For more details, please visit the webpage: https://www.globalmarketmonitor.com.cn/reports/2292280-hemoglobin-market-report.html.

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Supplementary Information

Supplementary Tables 1–10 and Supplementary Notes 1, 3 and 4.

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Supplementary Data

Supplementary Note 2. The sequence of all the oligonucleotides encoding crRNA.

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Chen, H., Wang, Y., Wang, W. et al. High-yield porphyrin production through metabolic engineering and biocatalysis. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02267-3

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