KR-102962127-B1 - Recombinant Bacillus strains with increased heme productivity and the method of producing heme using it

Abstract

The present invention relates to a recombinant Bacillus strain with increased heme productivity and a method for producing heme using the same. By confirming that heme productivity increases in a Bacillus strain transformed by introducing a recombinant plasmid containing a promoter of a heme-producing gene of the Bacillus strain and a hemA kk gene, the invention can be usefully utilized as a recombinant plasmid for heme production, a method for preparing a strain for heme production using the plasmid, and a method for producing heme using the strain.

Inventors

• 서승오
• 김근형
• 백광림

Assignees

• 가톨릭대학교 산학협력단

Dates

Publication Date

20260512

Application Date

20221027

Priority Date

20211029

Claims (6)

1. A recombinant plasmid for heme production comprising: a promoter of a gene related to heme production; and the Bacillus megaterium hemAkk gene represented by SEQ ID NO. 1 or the Bacillus subtilis hemAkk gene represented by SEQ ID NO. 2; The promoter of the above heme production-related gene is the CymR-PxylR expression cassette, and The above CymR-PxylR expression cassette is a recombinant plasmid having cumate-induced expression function.
2. delete
3. A method for producing a strain for heme production comprising the step of introducing the recombinant plasmid of claim 1 into a strain to obtain a transformant.
4. delete
5. A method for producing heme comprising the step of culturing the strain of claim 3.
6. delete

Description

Recombinant Bacillus strains with increased heme productivity and the method of producing heme using it The present invention relates to a recombinant Bacillus strain with increased heme productivity and a method for producing heme using the same. Porphyrins are representative tetradentate chelates widely found in living organisms, consisting of four linked pyrrole rings. This structure is called a porphin, and its derivatives are referred to as porphyrins. In plants, porphyrins are produced in forms similar to chlorophyll and play a vital role in life processes, earning them the nickname "the pigments of life." Various forms of porphyrin compounds exist in nature, and recently, they have been gaining attention as high-value compounds used as industrial materials, such as electrical conductors, pharmaceutical compounds, catalysts, and food ingredients. Within living organisms, heme is a representative porphyrin containing an iron atom in the center. Heme is a precursor to hemoglobin and possesses a porphyrin ring complexed with ferrous iron and protoporphyrin IX. In the human body, heme plays a crucial role in transporting oxygen and regulating intracellular pH, and due to its high absorption rate, it is utilized as an iron supplement. Recently, research into meat substitutes has been underway in the food industry due to environmental and ethical issues caused by excessive meat consumption and indiscriminate animal farming. Among these, research on alternative meats such as cultured meat, plant-based meat, and edible insects is receiving particular attention. Capturing the distinctive flavor, color, and texture of real meat is considered crucial for the commercialization and widespread adoption of plant-based meat. The blood flavor, which accounts for the majority of the taste of meat, is difficult to replicate from sources other than animal meat; therefore, finding a substitute that produces this blood flavor is essential for incorporating it into plant-based alternative meats. Based on this trend, heme is gaining attention for its role as an additive to enhance the taste and flavor of meat. However, existing heme production methods, which rely on extraction from animal blood and certain plant roots, have limitations as they are not only inefficient but also uneco-friendly. Consequently, if non-animal heme can be produced in an eco-friendly and sustainable manner by mass-producing these components through fermentation using microbial metabolic pathways, it could be utilized not only as an additive for plant-based alternative meats but also as a material for pharmaceutical and industrial applications. Figure 1 shows the heme biosynthetic metabolic pathway using the hemA kk gene in a Bacillus megaterium strain. Figure 2 shows the heme biosynthetic metabolic pathway using the hemA kk gene in a Bacillus subtilis strain. Figure 3 is a schematic diagram of the production of pIT5-hemA KK -Bm and pIT5-hemA KK -Bs plasmids. Figure 4 is a schematic diagram of introducing the hemA KK gene into a Bacillus megaterium strain using the pIT5-hemA KK -Bm plasmid. Figure 5 is a schematic diagram of introducing the hemA KK gene into a Bacillus subtilis strain using the pIT5-hemA KK -Bs plasmid. Figure 6 shows the results of colony PCR analysis of the introduction of the pIT5-hemA KK -Bm plasmid into a Bacillus megaterium strain. Figure 7 shows the results of colony PCR analysis of the introduction of the pIT5-hemA KK -Bs plasmid into a Bacillus subtilis strain. Figure 8 shows the results of comparing and analyzing the cell growth rates of wild-type and transformed Bacillus megaterium strains during 72 hours of flask culture. Figure 9 shows the results of comparing and analyzing the cell growth rates of wild-type and transformed Bacillus subtilis strains during 72 hours of flask culture. Figure 10 shows the results of comparing and analyzing the heme fermentation product production after 48 hours of wild-type and wild-type and transformed Bacillus megaterium strains. Figure 10A shows the wild-type (left) and transformed (right) strains, and Figure 10B shows the LB medium culture solution (left) and heme culture extract (right). Figure 11 shows the results of comparing and analyzing the production of heme fermentation products after 48 hours for wild-type and wild-type and transformed Bacillus subtilis strains. Figure 11A shows the wild-type (left) and transformed (right) strains, and Figure 11B shows the LB medium culture solution (left) and heme culture extract (right). Figure 12 shows the HPLC chromatogram results of heme (A) and CPGⅢ (B) 0.25 g/L standard substances. Figure 13 is a chromatogram result of HPLC-UV/Vis analysis of porphyrin production of wild-type and transformed Bacillus megaterium strains. Figure 14 is a chromatogram result of analyzing the porphyrin production of wild-type and transformed Bacillus subtilis strains using HPLC-UV/Vis. Figure 15 shows the results of comparing and analyzing the heme porphyrin