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Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF.

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Title: Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF.
Authors: Wu Y; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China., Sun Y; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China., Miao H; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China., Xia X; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China., Gao L; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.
Source: ACS synthetic biology [ACS Synth Biol] 2026 Aug 21; Vol. 15 (8), pp. 3272-3284.
Publication Type: Journal Article
Language: English
Journal Info: Publisher: American Chemical Society Country of Publication: United States NLM ID: 101575075 Publication Model: Print Cited Medium: Internet ISSN: 2161-5063 (Electronic) Linking ISSN: 21615063 NLM ISO Abbreviation: ACS Synth Biol Subsets: MEDLINE
Imprint Name(s): Original Publication: Washington, D.C. : American Chemical Society, c2012-
MeSH Terms: Bacillus subtilis*/metabolism , Bacillus subtilis*/genetics , Riboflavin*/metabolism , Flavin-Adenine Dinucleotide*/biosynthesis , Flavin-Adenine Dinucleotide*/metabolism , Membrane Transport Proteins*/metabolism , Membrane Transport Proteins*/genetics , Metabolic Engineering*/methods, Bacterial Proteins/metabolism ; Bacterial Proteins/genetics ; Streptomyces/genetics ; Streptomyces/metabolism ; Nucleotidyltransferases/metabolism ; Nucleotidyltransferases/genetics
Abstract: The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an "Uptake-Catalysis" system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADsP61W+R202Y, which increased catalytic efficiency (kcat/Km) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.
(© 2026 American Chemical Society.)
Grant Information: BK20233003 Basic Research Program of Jiangsu Province; 2023YFF1103703 National Key Research and Development Program of China
Contributed Indexing: Keywords: Bacillus subtilis; flavin adenine dinucleotide; isothermal compressibility perturbation engineering strategy; transporter
Substance Nomenclature: TLM2976OFR (Riboflavin)
146-14-5 (Flavin-Adenine Dinucleotide)
0 (Bacterial Proteins)
0 (Membrane Transport Proteins)
EC 2.7.7.- (Nucleotidyltransferases)
Entry Date(s): Date Created: 20260511 Date Completed: 20260822 Latest Revision: 20260822
Update Code: 20260823
DOI: 10.1021/acssynbio.6c00153
PMID: 42113034
Database: MEDLINE
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  Data: Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF.
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  Data: <searchLink fieldCode="AU" term="%22Wu+Y%22">Wu Y</searchLink>; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.<br /><searchLink fieldCode="AU" term="%22Sun+Y%22">Sun Y</searchLink>; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.<br /><searchLink fieldCode="AU" term="%22Miao+H%22">Miao H</searchLink>; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.<br /><searchLink fieldCode="AU" term="%22Xia+X%22">Xia X</searchLink>; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.<br /><searchLink fieldCode="AU" term="%22Gao+L%22">Gao L</searchLink>; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi214122, Jiangsu, China.
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  Data: <searchLink fieldCode="JN" term="%22101575075%22">ACS synthetic biology</searchLink> [ACS Synth Biol] 2026 Aug 21; Vol. 15 (8), pp. 3272-3284.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22American+Chemical+Society%22">American Chemical Society </searchLink><i>Country of Publication: </i>United States <i>NLM ID: </i>101575075 <i>Publication Model: </i>Print <i>Cited Medium: </i>Internet <i>ISSN: </i>2161-5063 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2221615063%22">21615063 </searchLink><i>NLM ISO Abbreviation: </i>ACS Synth Biol <i>Subsets: </i>MEDLINE
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  Data: <i>Original Publication</i>: Washington, D.C. : American Chemical Society, c2012-
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  Data: <searchLink fieldCode="MM" term="%22Bacillus+subtilis%22">Bacillus subtilis*</searchLink>/<searchLink fieldCode="MM" term="%22Bacillus+subtilis+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Bacillus+subtilis%22">Bacillus subtilis*</searchLink>/<searchLink fieldCode="MM" term="%22Bacillus+subtilis+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Riboflavin%22">Riboflavin*</searchLink>/<searchLink fieldCode="MM" term="%22Riboflavin+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Flavin-Adenine+Dinucleotide%22">Flavin-Adenine Dinucleotide*</searchLink>/<searchLink fieldCode="MM" term="%22Flavin-Adenine+Dinucleotide+biosynthesis%22">biosynthesis</searchLink> <br /><searchLink fieldCode="MM" term="%22Flavin-Adenine+Dinucleotide%22">Flavin-Adenine Dinucleotide*</searchLink>/<searchLink fieldCode="MM" term="%22Flavin-Adenine+Dinucleotide+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Membrane+Transport+Proteins%22">Membrane Transport Proteins*</searchLink>/<searchLink fieldCode="MM" term="%22Membrane+Transport+Proteins+metabolism%22">metabolism</searchLink> <br /><searchLink fieldCode="MM" term="%22Membrane+Transport+Proteins%22">Membrane Transport Proteins*</searchLink>/<searchLink fieldCode="MM" term="%22Membrane+Transport+Proteins+genetics%22">genetics</searchLink> <br /><searchLink fieldCode="MM" term="%22Metabolic+Engineering%22">Metabolic Engineering*</searchLink>/<searchLink fieldCode="MM" term="%22Metabolic+Engineering+methods%22">methods</searchLink><br /><searchLink fieldCode="MH" term="%22Bacterial+Proteins%22">Bacterial Proteins</searchLink>/<searchLink fieldCode="MH" term="%22Bacterial+Proteins+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Bacterial+Proteins%22">Bacterial Proteins</searchLink>/<searchLink fieldCode="MH" term="%22Bacterial+Proteins+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Streptomyces%22">Streptomyces</searchLink>/<searchLink fieldCode="MH" term="%22Streptomyces+genetics%22">genetics</searchLink> ; <searchLink fieldCode="MH" term="%22Streptomyces%22">Streptomyces</searchLink>/<searchLink fieldCode="MH" term="%22Streptomyces+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Nucleotidyltransferases%22">Nucleotidyltransferases</searchLink>/<searchLink fieldCode="MH" term="%22Nucleotidyltransferases+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Nucleotidyltransferases%22">Nucleotidyltransferases</searchLink>/<searchLink fieldCode="MH" term="%22Nucleotidyltransferases+genetics%22">genetics</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: The biosynthesis of flavin adenine dinucleotide (FAD), an essential redox coenzyme, is limited by inefficient substrate utilization and low catalytic activity. To address this, an "Uptake-Catalysis" system was constructed in Bacillus subtilis for FAD synthesis from riboflavin (RF). First, the Streptomyces davawensis transporter (RibM) was coexpressed with native FAD synthase (BsFADs) at a 2:1 ratio, which increased RF consumption to 51%. Subsequently, the isothermal compressibility perturbation engineering (ICPE) strategy was adopted, generating the optimum mutant BsFADsP61W+R202Y, which increased catalytic efficiency (kcat/Km) toward RF and FMN by 83% and 73%, respectively. Mechanistic analysis revealed that this improvement was due to a 39% shortening of RF substrate channel. Finally, FAD titer peaked at 563 mg/L with a conversion rate (cr-FAD) of 92% in shaking-flask fermentation optimization. This work achieved a 225-fold increase in FAD production, validating the effectiveness of the uptake-catalysis synergistic strategy, thereby establishing an efficient and scalable synthesis paradigm.<br /> (© 2026 American Chemical Society.)
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  Data: BK20233003 Basic Research Program of Jiangsu Province; 2023YFF1103703 National Key Research and Development Program of China
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  Data: <i>Keywords: </i>Bacillus subtilis; flavin adenine dinucleotide; isothermal compressibility perturbation engineering strategy; transporter
– Name: NumberCAS
  Label: Substance Nomenclature
  Group: ID
  Data: TLM2976OFR (Riboflavin)<br />146-14-5 (Flavin-Adenine Dinucleotide)<br />0 (Bacterial Proteins)<br />0 (Membrane Transport Proteins)<br />EC 2.7.7.- (Nucleotidyltransferases)
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  Data: <i>Date Created: </i>20260511 <i>Date Completed: </i>20260822 <i>Latest Revision: </i>20260822
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  Data: 10.1021/acssynbio.6c00153
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  Data: 42113034
PLink https://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=cmedm&AN=42113034
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        Value: 10.1021/acssynbio.6c00153
    Languages:
      – Code: eng
        Text: English
    PhysicalDescription:
      Pagination:
        StartPage: 3272
    Subjects:
      – SubjectFull: Bacterial Proteins metabolism
        Type: general
      – SubjectFull: Bacterial Proteins genetics
        Type: general
      – SubjectFull: Streptomyces genetics
        Type: general
      – SubjectFull: Streptomyces metabolism
        Type: general
      – SubjectFull: Nucleotidyltransferases metabolism
        Type: general
      – SubjectFull: Nucleotidyltransferases genetics
        Type: general
      – SubjectFull: Bacillus subtilis metabolism
        Type: general
      – SubjectFull: Bacillus subtilis genetics
        Type: general
      – SubjectFull: Riboflavin metabolism
        Type: general
      – SubjectFull: Flavin-Adenine Dinucleotide biosynthesis
        Type: general
      – SubjectFull: Flavin-Adenine Dinucleotide metabolism
        Type: general
      – SubjectFull: Membrane Transport Proteins metabolism
        Type: general
      – SubjectFull: Membrane Transport Proteins genetics
        Type: general
      – SubjectFull: Metabolic Engineering methods
        Type: general
    Titles:
      – TitleFull: Engineering of Riboflavin Transporter and Enzymes in Bacillus subtilis Facilitates High-Efficiency FAD Synthesis from RF.
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              M: 08
              Text: 2026 Aug 21
              Type: published
              Y: 2026
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