Heme has a variety of physiological functions in vivo and has been widely used in food, medicine, and other fields. Compared with traditional extraction methods, microbial synthesis of heme has the advantages of low cost and environmental friendliness. In this study, we constructed a co-culture system for heme biosynthesis by engineering the 5-aminolevulinic acid (5-ALA) synthesis module and the heme synthesis from the 5-ALA module into Bacillus amyloliquefaciens NX-2S154 with high glutamate anabolic flux and Escherichia coli BL21 with high expression ability of heterologous enzymes, respectively. Firstly, the polyglutamic acid synthase gene pgsBCA in B. amyloliquefaciens was knocked out by CRISPR-cas9n, and the fermentation results showed that B. amyloliquefaciens NX-2S154 had high intracellular glutamate synthesis flux, which indicated that it could provide sufficient glutamate precursors for the co-culture system of heme synthesis. Secondly, we divided the heme synthesis pathway into the precursor 5-ALA synthesis module and the module for heme synthesis from 5-ALA, which were constructed in B. amyloliquefaciens NX-2S154 and E. coli BL21, respectively, to obtain engineered strains B. amy-1 and E. coli-a. Co-cultivation of engineered strains B.amy-1 and E.coli-a resulted in a heme accumulation of 7.84 mg/L. Thirdly, the precursor 5-ALA synthesis module in B. amyloliquefaciens (B. amy-1) was further optimized to improve the efficiency of central carbon flux to heme through adaptively optimizing key pathway genes gltX, hemA ,and hemL, and knocking out ldh, pta, nas and prob genes to block the biosynthesis of by-products lactate, acetate, N-acetylaminoglutamate and proline. The above strategies resulted in the titer of heme in the co-culture system reaching 18.16 mg/L, which was 131.62% higher than that of the initial co-culture system. In addition, the 5-ALA extracellular transport pathway and the heme extracellular transport pathway were constructed to improve the synthesis efficiency of heme in the co-culture system and obtained 28.36 mg/L of heme. Finally, combining these strategies and further optimizing the fermentation process, we obtained a heme titer of 65.38 mg/L and a substrate molar conversion rate of 0.000382 mol/mol (heme/glucose) in a 7.5 L fermenter. The substrate conversion rate of the heme co-culture system constructed in this study was significantly higher than that of traditional single bacterial fermentation. These results also indicated that the co-culture strategy used in this study has great potential in constructing efficient cell factories to biosynthesize complex natural products.
PAN Fei
,
ZHU Yifan
,
YAN Yifan
,
DU Shanshan
,
LI Sha
,
XU Hong
,
LUO Zhengshan
. Construction and optimization of the co-culture system of Bacillus amyloliquefaciens and Escherichia coli to synthesize heme[J]. Food and Fermentation Industries, 2023
, 49(1)
: 1
-9
.
DOI: 10.13995/j.cnki.11-1802/ts.031446
[1] BRYANT D A, HUNTER C N, WARREN M J.Biosynthesis of the modified tetrapyrroles:The pigments of life[J].Journal of Biological Chemistry, 2020, 295(20):6 888-6 925.
[2] PERNER J, HATALOVA T, CABELLO D M, et al.Haem-responsive gene transporter enables mobilization of host haem in ticks[J].Open Biology, 2021, 11(9):210048.
[3] ZAMARREÑO BEAS J, VIDEIRA M A M, SARAIVA L M.Regulation of bacterial haem biosynthesis[J].Coordination Chemistry Reviews, 2022, 452:214286.
[4] 潘斐, 严一凡, 朱逸凡, 等.四吡咯化合物生物合成研究进展[J].生物工程学报, 2022, 38(4):1 307-1 321.
PAN F, YAN Y F, ZHU Y F, et al.Advances in the biosynthesis of tetrapyrrole compounds[J].Chinese Journal of Biotechnology, 2022, 38(4):1 307-1 321.
[5] QIU Y B, ZHU Y F, SHA Y Y, et al.Development of a robust Bacillus amyloliquefaciens cell factory for efficient poly(γ-glutamic acid) production from jerusalem artichoke[J].ACS Sustainable Chemistry & Engineering, 2020, 8(26):9 763-9 774.
[6] LUO Z S, LIU S, DU G C, et al.Enhanced pyruvate production in Candida glabrata by carrier engineering[J].Biotechnology and Bioengineering, 2018, 115(2):473-482.
[7] 曾娇娇, 余世琴, 周景文.代谢工程改造大肠杆菌增产酪醇[J].食品与发酵工业, 2021, 47(22):8-15.
ZENG J J, YU S Q, ZHOU J W.Metabolic engineering of Escherichia coli for improving tyrosol production[J].Food and Fermentation Industries, 2021, 47(22):8-15.
[8] KWON S J, DE BOER A L, PETRI R, et al.High-level production of porphyrins in metabolically engineered Escherichia coli:Systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis[J].Applied and Environmental Microbiology, 2003, 69(8):4 875-4 883.
[9] ZHAO X R, CHOI K R, LEE S Y.Metabolic engineering of Escherichia coli for secretory production of free haem[J].Nature Catalysis, 2018, 1(9):720-728.
[10] LI Z H, WANG X N, ZHANG H R.Balancing the non-linear rosmarinic acid biosynthetic pathway by modular co-culture engineering[J].Metabolic Engineering, 2019, 54:1-11.
[11] 张恕铭, 曾林, 孙向阳, 等.屎肠球菌与植物乳杆菌共培养产γ-氨基丁酸条件优化及关键酶活性研究[J].食品与发酵工业, 2021, 47(9):154-159.
ZHANG S M, ZENG L, SUN X Y, et al.Optimization of γ-aminobutyric acid produced by co-culturing Enterococcus faecium and Lactobacillus plantarum and the activities of key enzyme[J].Food and Fermentation Industries, 2021, 47(9):154-159.
[12] ZHOU K, QIAO K J, EDGAR S, et al.Distributing a metabolic pathway among a microbial consortium enhances production of natural products[J].Nature Biotechnology, 2015, 33(4):377-383.
[13] SHA Y Y, HUANG Y Y, ZHU Y F, et al.Efficient biosynthesis of low-molecular-weight poly-γ-glutamic acid based on stereochemistry regulation in Bacillus amyloliquefaciens[J].ACS Synthetic Biology, 2020, 9(6):1 395-1 405.
[14] SHA Y, ZHANG Y, QIU Y, et al.Efficient biosynthesis of low-molecular-weight poly-γ-glutamic acid by stable overexpression of PgdS hydrolase in Bacillus amyloliquefaciens NB[J].Journal of Agricultural and Food Chemistry, 2019, 67(1):282-290.
[15] QIU Y B, ZHU Y F, ZHANG Y T, et al.Characterization of a regulator pgsR on endogenous plasmid p2Sip and its complementation for poly(γ-glutamic acid) accumulation in Bacillus amyloliquefaciens[J].Journal of Agricultural and Food Chemistry, 2019, 67(13):3 711-3 722.
[16] QIU Y B, ZHANG Y T, ZHU Y F, et al.Improving poly-(γ-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing[J].Bioprocess and Biosystems Engineering, 2019, 42(10):1 711-1 720.
[17] TAN S I, YOU S C, SHIH I T, et al.Quantification, regulation and production of 5-aminolevulinic acid by green fluorescent protein in recombinant Escherichia coli[J].Journal of Bioscience and Bioengineering, 2020, 129(4):387-394.
[18] MISCEVIC D, MAO J Y, KEFALE T, et al.Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli[J].Biotechnology and Bioengineering, 2021, 118(1):30-42.
[19] NOH M H, LIM H G, PARK S, et al.Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli[J].Metabolic Engineering, 2017, 43:1-8.
[20] DONG Y S, ZHANG H, WANG X Y, et al.Enhancing ectoine production by recombinant Escherichia coli through step-wise fermentation optimization strategy based on kinetic analysis[J].Bioprocess and Biosystems Engineering, 2021, 44(7):1 557-1 566.
[21] CASTRO C C, NOBRE C, DE WEIRELD G, et al.Microbial co-culturing strategies for fructo-oligosaccharide production[J].New Biotechnology, 2019, 51:1-7.
[22] GOERS L, FREEMONT P, POLIZZI K M.Co-culture systems and technologies:Taking synthetic biology to the next level[J].Journal of the Royal Society, Interface, 2014, 11(96):20140065.
