Next Article in Journal
Role of Genomic, Economic, and Demographic Disparities in Mpox Epidemic in Africa: A Retrospective Cross-Country Analysis
Previous Article in Journal
Changes in the Gut Microbiota of Patients After SARS-CoV-2 Infection: What Do We Know?
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 13
Issue 11
10.3390/microorganisms13112530
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production

by
Jihua Zhao
1,†,
Teng Mu
1,†,
Dandan Niu
1,*,
Zhongzhen Ding
2,
Nokuthula Peace Mchunu
1,3,
Meng Zhang
1,
Suren Singh
4 and
Zhengxiang Wang
1,2,*
1
Research Center for Green Biomanufacturing, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China
2
Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
3
School of Life Science, University of KwaZulu Natal, Durban 4000, South Africa
4
Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, P. O. Box 1334, Durban 4001, South Africa
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(11), 2530; https://doi.org/10.3390/microorganisms13112530
Submission received: 8 September 2025 / Revised: 26 October 2025 / Accepted: 27 October 2025 / Published: 4 November 2025
(This article belongs to the Section Food Microbiology)
Browse Figures
Versions Notes

Abstract

Using Bacillus licheniformis H107 as the initial strain, a novel complementary synbiotics production method was developed through comprehensive metabolic engineering strategies. Key modifications included the systematic analysis and reconstruction of the central carbon metabolism pathway through precise gene editing, targeting the deletion of pflB, alsS, ydaP, and pycA genes while disrupting ganA1 and ganA2 to block galactooligosaccharide (GOS) metabolism. Additionally, heterologous expression of the L-lactate dehydrogenase gene (BcoaLDH) was implemented, resulting in the engineered strain H107-06A. Shake-flask fermentation under anaerobic conditions with 20 g/L glucose yielded L-lactic acid production of 4.45 g/L, representing a 15.3-fold increase compared to the wild type. In a 5 L fermenter using GOS syrup as the carbon source, the engineered system synergistically produced complementary synbiotics, comprising L-lactic acid (42.56 g/L), GOS (141.89 g/L, accounting for 75.09% of total sugars), and viable cells (3.82 g/L). These findings provide a foundation for developing innovative and functional fermentation products.

1. Introduction

The synergistic application of probiotics and prebiotics, collectively termed synbiotics, has garnered increasing attention for their potential to improve gut health and overall well-being [1,2,3]. Synbiotics stand out due to their unique ability to combine live microbial strains with functional substrates, conferring independent but complementary physiological activities [4,5]. Among prebiotics, galactooligosaccharides (GOS) exemplify functional efficacy thanks to their stability and exceptional physiochemical properties, enabling the selective promotion of beneficial gut bacteria [5,6,7]. However, current strategies for synbiotic production often rely heavily on the simple co-blending of probiotics and GOS, which lacks innovative integration or industrial scalability [2,8].
A novel scientific challenge emerges to overcome these limitations with biotechnologically advanced approaches capable of synergistically producing probiotics, GOS, and functional by-products through microbiological and metabolic engineering.
Commercially, GOS is traditionally produced through the enzymatic transglycosylation of lactose using β-galactosidase, during which glucose is unavoidably generated as a by-product [9]. This raises a pivotal question: can metabolic engineering be employed to construct specialized microbial strains capable of converting this glucose into valuable functional products, thereby developing a new generation of complementary synbiotics with enhanced functionality and economic feasibility?
Bacillus licheniformis, a widely recognized industrial microorganism, is characterized by high metabolic activity and a robust safety profile [10,11,12]. This species efficiently metabolizes simple sugars like glucose and offers the potential for producing functional products through metabolic engineering [13,14]. Among these, L-lactic acid stands out for its ability to lower intestinal pH and inhibit harmful bacterial growth [15,16,17]. Thus, constructing a strain with selective glucose metabolism and efficient L-lactic acid production could enable the combined production of high-purity GOS, viable cells, and functional metabolites. Nonetheless, the native B. licheniformis strain faces two major challenges: its intrinsic ability to degrade GOS, which prevents GOS retention, and its limited capacity for L-lactic acid synthesis due to the complex nature of pyruvate metabolism [18,19]. Genomic studies suggest that these challenges stem from β-galactosidase gene expression and multiple pyruvate metabolic branches. While single-gene manipulations have shown incremental improvements [20], the coordinated optimization of these pathways for efficient GOS retention and L-lactic acid production remains unexplored.
In this study, using metabolic engineering strategies, we systematically deleted the β-galactosidase genes and pyruvate metabolism branch genes in B. licheniformis H107 while introducing the exogenous L-lactate dehydrogenase gene, resulting in an engineered strain with selective metabolic and efficient L-lactic acid synthesis capabilities. This strain can selectively utilize the glucose in GOS syrup, converting it into L-lactic acid and viable cells while retaining GOS, achieving the synergistic production of three functional components. This process not only provides a novel preparation method for complementary synbiotics but also offers new ideas for innovative development in the field of fermentation engineering.

2. Materials and Methods

2.1. Strains, Vectors, Primers, and Chemicals

The strains and plasmids used in this study are listed in Table 1. Escherichia coli JM109 was used as the host cell for DNA manipulation and plasmid DNA preparation. B. licheniformis H107 was used as the wild type of the mutant strain in this study. Plasmid pUB-EX was a thermosensitive E. coli-Bacillus shuttle vector [21,22], which was utilized for markerless gene deletion in B. licheniformis. The plasmid pHY-P43-BcoaLDH cloned the encoding gene BcoaLDH of L-lactate dehydrogenase in Bacillus coagulans, which can enhance the synthesis and secretion of L-lactic acid in cells [23]. Cultivation of the strains was conducted at 37 °C and 220 rpm for 12–16 h in Luria–Bertani (LB) broth or LB plates at 37 °C. The media were supplemented with 100 µg/mL ampicillin or 20 µg/mL kanamycin when required. All oligonucleotide primers for gene amplification were synthesized by Shenggong Biotech (Shanghai) Co., Ltd., Shanghai, China; details are provided in Table S1. Galactooligosaccharide syrup was prepared in our laboratory with a concentration of 600 g/L, among which the glucose concentration was about 130 g/L [9].

2.2. Gene Deletion and Construction of Recombinant Bacteria

Plasmid DNA extraction, PCR amplification of DNA fragments, DNA recovery, restriction digestion, ligation, and identification of transformants were carried out according to standard genetic manipulation techniques [24]. Genetic transformation of B. licheniformis H107 was performed by electrotransformation as previously described [25]. The deletion of specific genes in B. licheniformis (pflB, alsS, ydaP, pycA, ganA1, and ganA2) was performed using homologous recombination techniques as described in prior studies [22]. Gene deletion experiments utilized upstream and downstream homologous arms of the target gene, amplified by PCR using genomic DNA from B. licheniformis H107. Deletion fragments, constructed via overlapping PCR, were cloned into the Sma I and Xba I sites of plasmid pUB-EX [21,22]. Recombinant plasmids were introduced into B. licheniformis through electroporation. Homologous recombination events were induced by cultivating transformants at elevated temperatures (42–45 °C), followed by screening for single- and double-crossover events to isolate mutant strains [21,22].

2.3. Flask Fermentation Experiments

The flask fermentation experiments were carried out according to the previous publications [26,27] in 250 mL flasks with the working volume of 50 mL. Briefly, cells were grown in 50 mL of LB medium in a 250 mL flask at 37 °C with shaking (200 rpm) for 10–12 h until a cell density (OD600) of 2.5–3.0 was reached. Cells were collected by centrifugation and resuspended in a modified M9 medium and then inoculated into 50 mL of the modified M9 medium complemented with 5 g/L glucose in a 250 mL flask with the initial cell density (OD600) of 0.15. For cell growth experiments, the cultivation was carried out at shaking speed of 200 rpm and at 37 °C. For lactate fermentation, the cultivation was first carried out at 37 °C and 200 rpm for 12 h, then 20 g/L glucose was added, followed by stationary cultivation (anaerobic fermentation) for lactate formation at 37 °C. Calcium carbonate with a final concentration of 40 g/L was added for neutralization. Sampling was carried out during the cultivation. Modified M9 medium contained (per liter) 15.11 g Na2HPO4·12H2O, 3 g KH2PO4, 1 g NH4Cl, and 0.5 g NaCl. A total of 1 mL of filter-sterilized 1 M MgSO4 and 1 mL of filter-sterilized trace element solution containing (per liter) 2.4 g FeCl3·6H2O, 0.3 g CoCl2·6H2O, 0.15 g CuCl2·2H2O, 0.3 g ZnCl2, 0.3g Na2MO4·2H2O, 0.075 g H3BO3, and 0.495 g MnCl2·4H2O was added to a liter of the final medium [26,27].

2.4. Bioreactor Experiments

A fed-batch fermentation experiment in a bioreactor was carried out according to the method described [27,28]. A 5 L bioreactor (Biotech-5bg; Baoxing Saisi Pharmaceutical Equipment (Shanghai) Co., Ltd., Shanghai, China), initially containing 2.2 L of the modified M9 medium as described above in the “Flask Fermentation Experiments” Section, was used for L-lactate production from GOS syrup. The two-phase fed-batch process was started by inoculating 200 mL fresh inoculum prepared by preculturing cells in fresh modified M9 medium as described above. The cells were cultivated in aerobic conditions followed by anaerobic fermentation. During the aerobic phase, GOS syrup was supplemented with 24 g/L for cell growth. The culture was grown at pH 6.5, controlled by automatically feeding 25% (w/v) NH4OH solution, and the dissolved oxygen tension was maintained at above 30% of saturation. Anaerobic fermentation for L-lactate formation was initiated by ceasing air sparging and reducing agitation to 200 rpm when the cell density (OD600) reached about 20. During the anaerobic phase, the pH was controlled at 6.5 by the addition of 25% (w/v) Ca(OH)2. The residual glucose concentration was maintained above 3 g/L by adding GOS syrup (980 mL of GOS syrup was added in total). The fermentations were stopped when the glucose was exhausted. The temperature during the aerobic culture stage of cells was 37 °C, and the temperature during the anaerobic fermentation stage was 42 °C.

2.5. Analytical Methods

Quantitative analysis of GOS, lactic acid, and related metabolites followed the methods previously described [9,26,28]. Glucose concentration was measured using a glucose biosensor (SBA-40C; Biology Institute of Shandong Academy of Sciences, Jinan, China). Samples were pretreated with H2SO4 (5% of sample volume) to release organic acids precipitated with CaCO3 or Ca(OH)2 during fermentation when necessary. Organic acids and ethanol were analyzed by a HPLC (Agilent 1260, Santa Clara, CA, USA) with UV (210 nm) and refractive index detectors using an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA, 300 × 7.8 mm) with 5 mmol/L H2SO4 as the eluent (0.6 mL/min; 65 °C; 20 μL injection). The standard curve ranged from 0.2 to 10.0 mg/mL using L-lactic acid, acetic acid, formic acid, and succinic acid standards with ≥98% purity (Sigma-Aldrich, Shanghai, China). Components were identified by comparing retention times with the standards, and contents were calculated by external standard methods. Lactic acid isomeric purity was determined using a chiral column (CLC-L; Advanced Separation Technologies, Whippany, NJ, USA) with 5 mM CuSO4 mobile phase (1 mL/min, 25 °C, 20 μL injection) and UV detection at 254 nm [29]. GOS quantification used a HPLC (Agilent 1260, Santa Clara, CA, USA) with RI detector and Sugar-Pak™ I column (Waters, Milford, MA, USA, 6.5 × 300 mm) with water as the mobile phase (0.5 mL/min, 80 °C, 20 μL injection). The standard curve ranged from 0.2 to 10.0 mg/mL using GOS standards (≥98% purity, Sigma-Aldrich). Each component was identified by comparing retention times with the standards, and content was calculated by external standard methods, expressed as percentage of dry weight.

2.6. Metabolic Network Analysis of B. licheniformis

Using the Bacillus licheniformis DSM13 genome as a reference, analysis was conducted according to the Biocyc Pathway (https://biocyc.org/) (accessed on 20 December 2020).

2.7. Data Statistics and Analysis

All experiments were repeated at least three times, and the experimental data were expressed as Mean ± standard deviation (Mean ± SD). Statistical analysis was performed using the SPSS 22.0 software. One-way ANOVA was used to evaluate the significance of differences among groups based on independent biological replicates. A p value < 0.05 indicated a statistically significant difference, and Tukey’s honestly significant difference test was employed as the post hoc test [30]. The chart drawing was completed using the Origin 2021 software.

3. Results

3.1. Metabolic Network Analysis of Pyruvate in B. licheniformis

Based on bioinformatics data, the central carbon metabolic network of strain H107 was reconstructed (Figure 1A). Pyruvate metabolism encompasses multiple branched pathways, including the PflB pathway (pyruvate → formic acid + acetyl-CoA), the AlsS pathway (pyruvate → acetolactate), the YdaP pathway (pyruvate → acetyl phosphate), and the PycA pathway (pyruvate → oxaloacetate). Guided by this analysis, sequential deletions of pflB, alsS, ydaP, and pycA genes were performed using homologous recombination techniques [21,22], verified by diagnostic PCR (Figure 1B). The resulting engineered strains were named H107-01 (ΔpflB), H107-02 (ΔpflB, ΔalsS), H107-03 (ΔpflB, ΔalsS, ΔydaP), and H107-04 (ΔpflB, ΔalsS, ΔydaP, ΔpycA).
The shake-flask fermentation results (Table 2) demonstrated the significant impact of these genetic modifications on metabolic distribution and function. Specifically, the deletion of pflB resulted in the complete inhibition of formic acid synthesis and a 79.3% increase in L-lactic acid production. Dual deletion of pflB and alsS further elevated lactic acid production by 13.5%, although acetate synthesis surged by 127.5% due to residual acetolactate pathways. Furthermore, ydaP deletion marginally affected acetic acid levels and improved L-lactic acid production, whereas pycA deletion sharply redirected carbon flow, increasing L-lactic acid output by 82.8% relative to H107-03. However, cumulative lactic acid production across these mutants remained suboptimal (0.29–1.06 g/L), necessitating further optimization to achieve industrially viable yields.

3.2. Selection of Lactose and GOS Non-Metabolizing Mutants

Bioinformatic analysis identified ganA1 and ganA2 as pivotal genes encoding β-galactosidases responsible for GOS and lactose metabolism in strain H107. These genes were sequentially deleted using homologous recombination techniques [21,22] from H107-04 to create H107-05 (ΔganA1) and H107-06 (ΔganA1, ΔganA2). Diagnostic PCR (Figure 2A) confirmed successful deletions, reducing the target gene sizes from 2.9 and 2.8 kb to approximately 1.2 kb each.
Carbon source utilization tests using glucose or GOS (20 g/L) as the sole carbon sources revealed that the mutant H107-06 retained its ability to grow efficiently in glucose (OD600: 3.25 ± 0.16 after 24 h) but completely lost its capacity to metabolize GOS (OD600: 0.12 ± 0.02). In contrast, its parental strain, H107-04, continued to grow normally in a GOS-based medium (OD600: 2.85 ± 0.14). This demonstrates that the dual deletion of ganA1 and ganA2 effectively blocks GOS degradation, enabling its selective retention for prebiotic functionality.

3.3. Enhancement of L-Lactic Acid Synthesis in B. licheniformis

To enhance the L-lactic acid synthesis capacity, the BcoaLDH gene encoding L-lactate dehydrogenase from B. coagulans was heterologously expressed in H107-06 using the recombinant plasmid pHY-P43-BcoaLDH (Lifeasible, Shirley, NY, USA). This construct features a strong P43 promoter for constitutive expression along with selection markers for Bacillus spp. [31].
Shake-flask fermentation revealed significant advancements: H107-06A (carrying pHY-P43-BcoaLDH) achieved an L-lactic acid yield of 4.45 ± 0.38 g/L (Table 3), representing a 222% improvement over H107-06 (1.06 ± 0.07 g/L). Kinetic evaluation showed a fourfold increase in lactic acid production efficiency, with minimal changes in glucose consumption rates, reflecting enhanced carbon conversion into L-lactic acid. The heterologous BcoaLDH expression likely improved the redox balance by providing an additional NADH oxidation pathway, thereby redirecting carbon flux from competing pathways toward L-lactic acid production. Additionally, the optical purity of the L-lactic acid produced reached up to 99.8%. This metabolic efficiency highlights the beneficial integration of exogenous BcoaLDH into B. licheniformis.

3.4. Establishment and Optimization of a Complementary Synbiotic Production Process

The synergistic production capability of strain H107-06A was validated in a 5 L fermenter using GOS syrup as the carbon source. This process was optimized to achieve distinct operational phases: an aerobic biomass accumulation phase (0–22 h), a microaerobic acid production phase (22–60 h), and a stationary phase (60–72 h), consistent with established fermentation strategies for maximizing product yields [13,32]. During the acid production phase, reduced agitation and controlled pH effectively redirected metabolic flux toward L-lactic acid synthesis, while minimizing by-product formation [10].
At the end of 72 h, H107-06A produced 42.56 ± 1.85 g/L of L-lactic acid with a productivity of 0.59 ± 0.03 g/(L·h) (Table 4). GOS retention reached 141.89 ± 3.56 g/L, accounting for 75.09 ± 1.85% of total sugars, and active biomass concentration was 3.82 ± 0.15 g/L. These results demonstrate a 14.7-fold improvement in lactic acid production compared to the wild-type strain and highlight the efficacy of the engineered system for simultaneous synergistic production of prebiotics, probiotics, and functional metabolites. This aligns with prior findings on the potential of microbial engineering to enable multifunctional fermentation technologies [33].
Following exploration and research into complementary synbiotic production processes, a next-generation process route for synbiotic development has been proposed (Figure 3). Lactose is enzymatically converted into a GOS syrup using a β-galactosidase with high transglycosylation activity [9]. This syrup serves as the carbon source for B. licheniformis fermentation. During this fermentation, the glucose by-product present in the GOS syrup is completely consumed, yielding L-lactic acid and active bacterial biomass, while simultaneously enhancing the purity of the GOS. This technology differs fundamentally from the conventional simple blending of probiotics with GOS. It leverages metabolic engineering technology to achieve the synergistic production of active bacterial cells, high-purity GOS, and functional L-lactic acid, thereby providing an innovative technological pathway for the development of complementary synbiotics.

4. Discussion

This study significantly enhanced L-lactic acid synthesis capabilities by systematically deleting pyruvate metabolic branch genes. Comprehensive genetic modifications were performed, including the deletion of pflB, alsS, ydaP, and pycA, to redirect carbon flux towards L-lactate production [28,34]. The deletion of pflB resulted in a near-complete suppression of formic acid synthesis, while increasing L-lactic acid output by 79% (from 0.29 g/L to 0.52 g/L). Sequential deletions of alsS and ydaP elevated L-lactate production to 0.58 g/L, albeit with increased acetate accumulation—a potential outcome of intracellular NADH/NAD+ balance regulation [32,35]. The final deletion of pycA redirected pyruvate flux away from the tricarboxylic acid cycle, yielding L-lactic acid levels of 1.06 g/L—a remarkable step towards industrially relevant production. These findings align with prior insights into the utility of pathway engineering for optimizing microbial metabolite synthesis [36,37].
Moreover, the successful double knockout of genes ganA1 and ganA2 effectively blocked GOS degradation pathways while preserving its prebiotic functionality. This innovative approach achieved the selective utilization of glucose in GOS syrup, enhancing substrate purity and fermentation efficiency [9]. The preservation of GOS is critical for synbiotic applications, as it directly contributes to the functional properties of the final product [1].
Anaerobic conditions significantly enhance L-lactic acid production, which is related to the accumulation of NADH and the increased activity of lactate dehydrogenase [38]. During the scale-up process in the fermenter, the L-lactic acid synthesis was further optimized by regulating dissolved oxygen levels and pH. Optimizing the feeding strategy effectively reduced substrate inhibition, raising the yield to 42.56 g/L. Research indicates that maintaining an appropriate glucose concentration (5–10 g/L) is beneficial in reducing by-product accumulation [39,40].

5. Study Limitations and Future Perspectives

Although the engineered B. licheniformis strain demonstrated promising results at the 5 L scale, several aspects merit further investigation. The metabolic stability of the recombinant strain during prolonged fermentation, possible plasmid loss under industrial conditions, and downstream purification challenges for high-purity GOS require assessment. Future studies could focus on chromosomal integration of BcoaLDH to enhance genetic stability and on multi-omics analyses (transcriptomics and metabolomics) to elucidate global regulatory mechanisms driving L-lactic acid accumulation and GOS retention. Co-culture strategies with the probiotic Lactobacillus or Bifidobacterium species may also enhance the synbiotic functionality of the final product.

6. Conclusions

This study successfully demonstrates the potential of metabolic engineering in B. licheniformis for developing innovative complementary synbiotics. By systematically engineering pyruvate metabolic pathways and blocking GOS degradation, the engineered strain H107-06A achieved remarkable improvements in L-lactic acid production (42.56 g/L in a 5 L fermenter), glucose conversion efficiency, and GOS retention (75.09%). These findings establish a scalable and synergistic fermentation platform for producing probiotics, prebiotics, and functional metabolites.
The study provides a scientifically robust framework for overcoming existing limitations in synbiotic production, contributing to advancements in the food and health industries and supporting broader applications in functional nutrition [1,5]. Future work may explore additional pathway optimizations or co-culture systems to further enhance metabolic efficiencies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13112530/s1, Table S1: The oligonucleotide sequence of primers used in this study.

Author Contributions

Conceptualization, D.N., S.S. and Z.W.; data curation, J.Z., T.M., D.N. and Z.W.; formal analysis, J.Z., T.M. and M.Z.; funding acquisition, D.N., N.P.M. and Z.W.; investigation, J.Z., T.M., Z.D. and D.N.; methodology, J.Z., T.M., D.N., M.Z. and Z.W.; project administration, D.N.; resources, D.N., N.P.M. and Z.W.; software, J.Z. and T.M.; supervision, D.N., S.S. and Z.W.; writing—original draft preparation, J.Z., T.M. and D.N.; writing—review and editing, J.Z., T.M., D.N., N.P.M., M.Z., S.S. and Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Tianjin Outstanding Talent Program (Grant No.: JC20200309) to Z.W. and the Intergovernmental International Scientific and Technological Innovation Cooperation Program, MOST, China, to D.N. (Grant No.: 2021YFE0106200) and to Z.W. (Grant No.: 2025YFE0101200).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors would like to express their sincere gratitude to Ling Dong from China National Research Institute of Food and Fermentation Industries Co., Ltd. for her valuable guidance, assistance, and support during the preparation of the research proposal, manuscript organization, and writing process.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mounir, M.; Ibijbijen, A.; Farih, K.; Rabetafika, H.N.; Razafindralambo, H.L. Synbiotics and their antioxidant properties, mechanisms, and benefits on human and animal health: A narrative review. Biomolecules 2022, 12, 1443. [Google Scholar] [CrossRef]
  2. Luzzi, A.; Briata, I.M.; Di Napoli, I.; Giugliano, S.; Di Sabatino, A.; Rescigno, M.; Cena, H. Prebiotics, probiotics, synbiotics and postbiotics to adolescents in metabolic syndrome. Clin. Nutr. 2024, 43, 1433–1446. [Google Scholar] [CrossRef] [PubMed]
  3. Khursheed, R.; Gulati, M.; Wadhwa, S.; Vishwas, S.; Sharma, D.S.; Corrie, L.; Alam, A.; Alnasser, S.M.; Aba Alkhayl, F.F.; Parveen, Z.; et al. Multifaceted role of synbiotics as nutraceuticals, therapeutics and carrier for drug delivery. Chem. Biol. Interact. 2022, 368, 110223. [Google Scholar] [CrossRef] [PubMed]
  4. Gomez Quintero, D.F.; Kok, C.R.; Hutkins, R. The future of synbiotics: Rational formulation and design. Front. Microbiol. 2022, 13, 919725. [Google Scholar] [CrossRef]
  5. Lee, S.; Choi, S.P.; Choi, H.J.; Jeong, H.; Park, Y.S. A comprehensive review of synbiotics: An emerging paradigm in health promotion and disease management. World J. Microbiol. Biotechnol. 2024, 40, 280. [Google Scholar] [CrossRef]
  6. Fukaya, M.; Yokoyama, Y.; Usui, H.; Fujieda, H.; Sakatoku, Y.; Takahashi, T.; Miyata, K.; Niikura, M.; Sugimoto, T.; Asahara, T.; et al. Impact of synbiotics treatment on bacteremia induced during neoadjuvant chemotherapy for esophageal cancer: A randomised controlled trial. Clin. Nutr. 2021, 40, 5781–5791. [Google Scholar] [CrossRef]
  7. Kanazawa, A.; Aida, M.; Yoshida, Y.; Kaga, H.; Katahira, T.; Suzuki, L.; Tamaki, S.; Sato, J.; Goto, H.; Azuma, K.; et al. Effects of synbiotic supplemen tation on chronic inflammation and the gut microbiota in obese patients with type 2 diabetes mellitus: A randomized controlled study. Nutrients 2021, 13, 558. [Google Scholar] [CrossRef]
  8. Swanson, K.S.; Gibson, G.R.; Hutkins, R.; Reimer, R.A.; Reid, G.; Verbeke, K.; Scott, K.P.; Holscher, H.D.; Azad, M.B.; Delzenne, N.M.; et al. The international scientific association for probiotics and prebiotics (ISAPP) consensus statement on the definition and scope of synbiotics. Nat. Rev. Gastroenterol. Hepatol. 2020, 17, 687–701. [Google Scholar] [CrossRef] [PubMed]
  9. Zhao, J.; Niu, D.; Liu, J.; Jin, Z.; Mchunu, N.P.; Singh, S.; Wang, Z. Enhancing β-galactosidase performance for galactooligosaccharides preparation via strategic glucose re-tunneling. Int. J. Mol. Sci. 2024, 25, 12316. [Google Scholar] [CrossRef]
  10. Muras, A.; Romero, M.; Mayer, C.; Otero, A. Biotechnological applications of Bacillus licheniformis. Crit. Rev. Biotechnol. 2021, 41, 609–627. [Google Scholar] [CrossRef]
  11. He, H.; Yu, Q.; Ding, Z.; Zhang, L.; Shi, G.; Li, Y. Biotechnological and food synthetic biology potential of platform strain: Bacillus licheniformis. Synth. Syst. Biotechnol. 2023, 8, 281–291. [Google Scholar] [CrossRef] [PubMed]
  12. Dai, H.; Ma, L.; Xu, Z.; Soteyome, T.; Yuan, L.; Yang, Z.; Jiao, X.A. Invited review: Role of Bacillus licheniformis in the dairy industry-Friends or foes? J. Dairy Sci. 2024, 107, 7520–7532. [Google Scholar] [CrossRef] [PubMed]
  13. Zhang, Z.; He, P.; Hu, S.; Yu, Y.; Wang, X.; Ishaq, A.R.; Chen, S. Promoting cell growth for biochemicals production via boosting the synthesis of L/D-alanine and D-alanyl-D-alanine in Bacillus licheniformis. World J. Microbiol. Biotechnol. 2023, 39, 115. [Google Scholar] [CrossRef]
  14. Wang, C.; Niu, D.; Zhou, Y.; Liu, H.; Mchunu, N.P.; Zhang, M.; Singh, S.; Wang, Z. Synergistic engineering of the twin-arginine translocation (Tat) pathway and membrane capacity enhances extracellular production of amylosucrase in Bacillus licheniformis. Microorganisms 2025, 13, 1179. [Google Scholar] [CrossRef]
  15. Hofvendahl, K.; Hahn-Hägerdal, B. Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb. Technol. 2000, 26, 87–107. [Google Scholar] [CrossRef]
  16. Sun, S.; Li, H.; Chen, J.; Qian, Q. Lactic Acid: No Longer an inert and end-product of glycolysis. Physiology 2017, 32, 453–463. [Google Scholar] [CrossRef]
  17. Bilal, M.; Niu, D.; Wang, Z. Novel enzyme-fermentation process for bioconversion of restaurant food waste into isomaltooligosaccharide-and L-lactic acid-enriched animal feed. Front. Sustain. Food Syst. 2024, 8, 1326304. [Google Scholar] [CrossRef]
  18. Elsayed, E.A.; Danial, E.N.; Wadaan, M.A.; El-Enshasy, H.A. Production of β-galactosidase in shake-flask and stirred tank bioreactor cultivations by a newly isolated Bacillus licheniformis strain. Biocatal. Agric. Biotechnol. 2019, 20, 101231. [Google Scholar] [CrossRef]
  19. Ren, Z.Q.; Liu, L.H.; Tang, T.X.; Huang, K.; Huang, Z. Effectively increase the L(+)-isomer proportion of ethyl lactate in Baijiu by isolating and applying L(+)-lactic acid-producing bacteria. Food Biosci. 2025, 63, 105615. [Google Scholar] [CrossRef]
  20. Li, B.; Cai, D.; Chen, S. Metabolic engineering of central carbon metabolism of Bacillus licheniformis for enhanced production of poly-γ-glutamic acid. Appl. Biochem. Biotechnol. 2021, 193, 3540–3552. [Google Scholar] [CrossRef]
  21. Shen, P.; Niu, D.; Liu, X.; Tian, K.; Permaul, K.; Singh, S.; McHunu, N.P.; Wang, Z. High-efficiency chromosomal integrativeamplification strategy for overexpressing alpha-amylase in Bacillus licheniformis. J. Ind. Microbiol. Biotechnol. 2022, 49, kuac009. [Google Scholar] [CrossRef]
  22. Wang, C.; Niu, D.; McHunu, N.P.; Zhang, M.; Singh, S.; Wang, Z. Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway. J. Ind. Microbiol. Biotechnol. 2024, 51, kuae004. [Google Scholar] [CrossRef] [PubMed]
  23. Tian, K.; Shi, G.; Lu, F.; Singh, S.; Wang, Z. High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli. Chin. J. Biotechnol. 2013, 29, 1268–1277. [Google Scholar]
  24. Sambrook, J.; Russel, D. Molecular Cloning: A Laboratory Manual, 3rd ed.; Cold Spring Harbor Press: Woodbury, NY, USA, 2001. [Google Scholar]
  25. Xu, M.; Ma, J.; Wang, Z. Effect of high osmolarity on electro-transformation efficiency of bacteria. J. Wuxi Uni. Light Ind. 2004, 23, 98–100. [Google Scholar]
  26. Zhou, L.; Zuo, Z.; Chen, X.; Niu, D.; Tian, K.; Prior, B.A.; Shen, W.; Shi, G.; Singh, S.; Wang, Z. Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli. Curr. Microbiol. 2011, 62, 981–989. [Google Scholar] [CrossRef]
  27. Zhou, L.; Niu, D.; Tian, K.; Chen, X.; Prior, B.A.; Shen, W.; Shi, G.; Singh, S.; Wang, Z. Genetically switched D-lactate production in Escherichia coli. Metab. Eng. 2012, 14, 560–568. [Google Scholar] [CrossRef]
  28. Niu, D.; Tian, K.; Prior, B.A.; Wang, M.; Wang, Z.; Lu, F.; Singh, S. Highly efficient L-lactate production using engineered Escherichia coli with dissimilar temperature optima for L-lactate formation and cell growth. Microb. Cell Fact. 2014, 13, 78. [Google Scholar] [CrossRef]
  29. Wang, M.; Niu, D.; Gao, M.; Wang, A.; Zhao, W.; Permaul, K.; Singh, S.; Wang, Z. Decoupling sucrose utilization from oxygen-responsive regulation for high-efficiency L-lactic acid production in Escherichia coli. Biotechnol. Biofuels Bioprod. 2025, 18, 101. [Google Scholar] [CrossRef] [PubMed]
  30. Das, A.; Prakash, G.; Lali, A.M. 2,3-Butanediol production using soy-based nitrogen source and fermentation process evaluation by a novel isolate of Bacillus licheniformis BL1. Prep. Biochem. Biotechnol. 2021, 51, 1046–1055. [Google Scholar] [CrossRef]
  31. Niu, D.; Wang, Z. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis. J. Ind. Microbiol. Biotechnol. 2007, 34, 357–362. [Google Scholar] [CrossRef]
  32. Wang, Y.; Sun, K.; Sheng, F.; Zhao, B.; Wu, Y.; Jin, Z.; Wang, J.; Li, S. Metabolic engineering of Bacillus licheniformis for the efficient production of D-lactate from hemicellulose. Biotechnol. Bioeng. 2022, 119, 1679–1690. [Google Scholar] [CrossRef]
  33. Abedin, M.M.; Chourasia, R.; Phukon, L.C.; Sarkar, P.; Ray, R.C.; Singh, S.P.; Rai, A.K. Lactic acid bacteria in the functional food industry: Biotechnological properties and potential applications. Crit. Rev. Food Sci. Nutr. 2024, 64, 10730–10748. [Google Scholar] [CrossRef] [PubMed]
  34. Cao, J.; Yu, Z.; Liu, W.; Zhao, J.; Zhang, H.; Zhai, Q.; Chen, W. Probiotic characteristics of Bacillus coagulans and associated implications for human health and diseases. J. Funct. Foods 2020, 64, 103643. [Google Scholar] [CrossRef]
  35. Wang, X.; Pan, J.; Wu, J.; Chen, X.; Gao, C.; Song, W.; Wei, W.; Liu, J.; Liu, L. Regulation of intracellular level of ATP and NADH in Escherichia coli to promote succinic acid production. Chin. J. Biotechnol. 2023, 39, 3236–3252. [Google Scholar] [CrossRef]
  36. Yin, X.; Li, J.; Shin, H.D.; Du, G.; Liu, L.; Chen, J. Metabolic engineering in the biotechnological production of organic acids in the tricarboxylic acid cycle of microorganisms: Advances and prospects. Biotechnol. Adv. 2015, 33, 830–841. [Google Scholar] [CrossRef]
  37. Zhang, S.; Wakai, S.; Sasakura, N.; Tsutsumi, H.; Hata, Y.; Ogino, C.; Kondo, A. Pyruvate metabolism redirection for biological production of commodity chemicals in aerobic fungus Aspergillus oryzae. Metab. Eng. 2020, 61, 225–237. [Google Scholar] [CrossRef]
  38. Chang, D.E.; Jung, H.C.; Rhee, J.S.; Pan, J.G. Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1. Appl. Environ. Microbiol. 1999, 65, 1384–1389. [Google Scholar] [CrossRef]
  39. Xu, X.; Wang, H.; Chen, X.; Wu, J.; Gao, C.; Song, W.; Wei, W.; Liu, J.; Liu, Y.; Liu, L. Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production. Chin. J. Biotechnol. 2024, 40, 2513–2527. [Google Scholar] [CrossRef]
  40. Xu, J.Z.; Yu, H.B.; Han, M.; Liu, L.M.; Zhang, W.G. Metabolic engineering of glucose uptake systems in corynebacterium glutamicum for improving the efficiency of L-lysine production. J. Ind. Microbiol. Biotechnol. 2019, 46, 937–949. [Google Scholar] [CrossRef]
Microorganisms 13 02530 g001
Figure 1. Pyruvate metabolic network and regulation in B. licheniformis. (A): Analysis of pyruvate metabolic pathways in B. licheniformis. (B): Deletions of pflB, alsS, ydaP, and pycA in B. licheniformis H107 and diagnostic PCR verification of mutant strains. M: 1 kb DNA Marker; Lanes 1–2: mutations of pflB in mutant H107-01; Lanes 3–6: mutations of pflB and alsS simultaneously occurred in mutant H107-02; Lanes 7–12: mutations of pflB, alsS, and ydaP occurred simultaneously in mutant H107-03; and Lanes 13–20: mutations of pflB, alsS, ydaP, and pycA occurred simultaneously.
Figure 1. Pyruvate metabolic network and regulation in B. licheniformis. (A): Analysis of pyruvate metabolic pathways in B. licheniformis. (B): Deletions of pflB, alsS, ydaP, and pycA in B. licheniformis H107 and diagnostic PCR verification of mutant strains. M: 1 kb DNA Marker; Lanes 1–2: mutations of pflB in mutant H107-01; Lanes 3–6: mutations of pflB and alsS simultaneously occurred in mutant H107-02; Lanes 7–12: mutations of pflB, alsS, and ydaP occurred simultaneously in mutant H107-03; and Lanes 13–20: mutations of pflB, alsS, ydaP, and pycA occurred simultaneously.
Microorganisms 13 02530 g001
Microorganisms 13 02530 g002
Figure 2. Creation of mutant strain H107-06 and its carbon source utilization characteristics. (A): Deletion of ganA1 and ganA2 in mutant strain H107-04 and diagnostic PCR verification of the mutant strains; M: 1 kb DNA Marker; lanes 1–10: mutations of pflB, alsS, ydaP, pycA, and ganA1 occurring simultaneously in mutant strain H107-05; lanes 11–22: mutations of pflB, alsS, ydaP, pycA, ganA1, and ganA2 occurring simultaneously in mutant strain H107-06. (B): Growth characteristics of mutant strains under shake-flask culture.
Figure 2. Creation of mutant strain H107-06 and its carbon source utilization characteristics. (A): Deletion of ganA1 and ganA2 in mutant strain H107-04 and diagnostic PCR verification of the mutant strains; M: 1 kb DNA Marker; lanes 1–10: mutations of pflB, alsS, ydaP, pycA, and ganA1 occurring simultaneously in mutant strain H107-05; lanes 11–22: mutations of pflB, alsS, ydaP, pycA, ganA1, and ganA2 occurring simultaneously in mutant strain H107-06. (B): Growth characteristics of mutant strains under shake-flask culture.
Microorganisms 13 02530 g002
Microorganisms 13 02530 g003
Figure 3. An integrated framework for new complementary synbiotic development. The left and right panels illustrate conventional methods for producing complementary synbiotics, which involves the separate preparation of probiotics and prebiotics prior to mixing. The central panel highlights our innovative approach using metabolically engineered B. licheniformis for simultaneous high-yield L-lactic acid production while preserving galactooligosaccharide integrity, enabling efficient complementary synbiotic production in a single integrated process.
Figure 3. An integrated framework for new complementary synbiotic development. The left and right panels illustrate conventional methods for producing complementary synbiotics, which involves the separate preparation of probiotics and prebiotics prior to mixing. The central panel highlights our innovative approach using metabolically engineered B. licheniformis for simultaneous high-yield L-lactic acid production while preserving galactooligosaccharide integrity, enabling efficient complementary synbiotic production in a single integrated process.
Microorganisms 13 02530 g003
Table 1. Strains and plasmids used in this study.
Table 1. Strains and plasmids used in this study.
Strains/VectorsCharacteristicsSource
Strains
E. coli JM109endA1, recA1, gyrA96, thi, hsdR17, relA1, supE44, λ-, Δ(lac-proAB), [F’, traD36, proAB, laqIqZΔM15]Lab stock
B. licheniformis H107Wild-type B. licheniformisLab stock
B. licheniformis H107-01∆pflBThis study
B. licheniformis H107-02∆pflB, ∆alsSThis study
B. licheniformis H107-03∆pflB, ∆alsS, ∆ydaPThis study
B. licheniformis H107-04∆pflB, ∆alsS, ∆ydaP, ∆pycA,This study
B. licheniformis H107-04A∆pflB, ∆alsS, ∆ydaP, ∆pycA, pHY-P43-BcoaLDHThis study
B. licheniformis H107-05∆pflB, ∆alsS, ∆ydaP, ∆pycA, ∆ganA2This study
B. licheniformis H107-06∆pflB, ∆alsS, ∆ydaP, ∆pycA, ∆ganA2, ∆ganA1This study
B. licheniformis H107-06A∆pflB, ∆alsS, ∆ydaP, ∆pycA, ∆ganA2, ∆ganA1, pHY-P43-BcoaLDHThis study
Vectors
pUB-EXKmR, Derived from T2(2)-ori, E. coli-Bacillus shµttle vector, thermosensitive replication[21,22]
pUB-EX-pflBKmR, thermosensitive plasmid, used for deletion of pflBThis study
pUB-EX-alsSKmR, thermosensitive plasmid, used for deletion of alsSThis study
pUB-EX-ydaPKmR, thermosensitive plasmid, used for deletion of ydaPThis study
pUB-EX-pycAKmR, thermosensitive plasmid, used for deletion of pycAThis study
pUB-EX-ganA2KmR, thermosensitive plasmid, used for deletion of ganA2Lab stock
pUB-EX-ganA1KmR, thermosensitive plasmid, used for deletion of ganA1Lab stock
pHY-P43-BcoaLDHTetR, AmpR, P43, BcoaLDH[23]
Table 2. Distribution of major metabolites in each mutant strain (shake-flask level).
Table 2. Distribution of major metabolites in each mutant strain (shake-flask level).
StrainsL-Lactic Acid
(g·L−1)		Acetic Acid
(g·L−1)		Formic Acid
(g·L−1)		Succinic Acid
(g·L−1)		Biomass
(g·L−1)
WT0.29 ± 0.030.54 ± 0.030.63 ± 0.123.75 ± 0.281.52 ± 0.11
H107-010.52 ± 0.04 *0.40 ± 0.02 *ND2.85 ± 0.21 *1.43 ± 0.09
H107-020.59 ± 0.04 *0.91 ± 0.06 **ND2.66 ± 0.19 *1.38 ± 0.10
H107-030.58 ± 0.03 **0.85 ± 0.06 **ND2.84 ± 0.21 *1.35 ± 0.08
H107-041.06 ± 0.07 **0.70 ± 0.05 **ND3.01 ± 0.22 *1.31 ± 0.09
ND indicates not detectable. * p < 0.05 and ** p < 0.01, compared with WT. Fermentation conditions: 20 g/L glucose, 37 °C, and 48 h.
Table 3. L-lactic acid fermentation performance of H107-06A under different conditions.
Table 3. L-lactic acid fermentation performance of H107-06A under different conditions.
Cultivation ConditionsL-Lactic Acid
(g·L−1)		Glucose Consumption
(g·L−1)		Conversion Rate
(%)		Biomass
(g·L−1)
Aerobic2.52 ± 0.238.64 ± 0.4629.15 ± 1.232.13 ± 0.15
Microaerobic3.24 ± 0.319.43 ± 0.4934.37 ± 1.671.82 ± 0.12
Anaerobic4.45 ± 0.389.90 ± 0.5244.93 ± 1.941.57 ± 0.11
The data was the mean ± standard deviation (n = 3). Conversion rate = L-lactic acid production/Glucose consumption ×100%. Fermentation conditions: 20 g/L glucose, 37 °C and 48 h.
Table 4. Preparation and comparison of novel complementary synbiotic by strain H107-06A in a 5 L fermenter.
Table 4. Preparation and comparison of novel complementary synbiotic by strain H107-06A in a 5 L fermenter.
IndicatorWild TypeH107-06AFold Increase
L-Lactic acid (g·L−1)2.89 ± 0.4542.56 ± 1.85 ***14.7
Glucose conversion rate (%)25.38 ± 1.8685.12 ± 2.35 ***4.4
Biomass (g·L−1)2.55 ± 0.183.82 ± 0.15 ***1.5
Relative purity of GOS (%)53.67 ± 2.1275.09 ± 1.85 ***1.4
*** p < 0.001, compared with WT.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Zhao, J.; Mu, T.; Niu, D.; Ding, Z.; Mchunu, N.P.; Zhang, M.; Singh, S.; Wang, Z. Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production. Microorganisms 2025, 13, 2530. https://doi.org/10.3390/microorganisms13112530

AMA Style

Zhao J, Mu T, Niu D, Ding Z, Mchunu NP, Zhang M, Singh S, Wang Z. Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production. Microorganisms. 2025; 13(11):2530. https://doi.org/10.3390/microorganisms13112530

Chicago/Turabian Style

Zhao, Jihua, Teng Mu, Dandan Niu, Zhongzhen Ding, Nokuthula Peace Mchunu, Meng Zhang, Suren Singh, and Zhengxiang Wang. 2025. "Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production" Microorganisms 13, no. 11: 2530. https://doi.org/10.3390/microorganisms13112530

APA Style

Zhao, J., Mu, T., Niu, D., Ding, Z., Mchunu, N. P., Zhang, M., Singh, S., & Wang, Z. (2025). Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production. Microorganisms, 13(11), 2530. https://doi.org/10.3390/microorganisms13112530

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop