Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris.

Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using PAOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

[Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7].

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Multilayer Genetic Circuits for Dynamic Regulation of Metabolic Pathways.

The dynamic regulation of metabolic pathways is based on changes in external signals and endogenous changes in gene expression levels and has extensive applications in the field of synthetic biology and metabolic engineering. However, achieving dynamic control is not trivial, and dynamic control is difficult to obtain using simple, single-level, control strategies because they are often affected by native regulatory networks. Therefore, synthetic biologists usually apply the concept of logic gates to build more complex and multilayer genetic circuits that can process various signals and direct the metabolic flux toward the synthesis of the molecules of interest. In this review, we first summarize the applications of dynamic regulatory systems and genetic circuits and then discuss how to design multilayer genetic circuits to achieve the optimal control of metabolic fluxes in living cells.

[Strategies and tools for metabolic engineering in Bacillus subtilis].

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.

Engineering a ComA Quorum-Sensing circuit to dynamically control the production of Menaquinone-4 in Bacillus subtilis.

Menaquinone-4 (MK-4) plays a significant role in bone health and cardiovascular therapy. Although many strategies have been adopted to increase the yield of MK-4 in Bacillus subtilis 168, the effectiveness of MK-4 is still low due to the inherent limitations of metabolic pathways. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. Nevertheless, in most reports, QS systems depend on down-regulated expression rather than up-regulated expression, which greatly limit their potential as molecular switches to control metabolic flux. To address this challenge, a modular PhrQ-RapQ-ComA QS system is developed based on promoter PA11, which is up-regulated by phosphorylated ComA (ComA-P). In this paper, firstly we analyzed the ComA-based gene expression regulation system in Bacillus subtilis 168. We constructed a promoter library of diff ;erent abilities, selected best promoters from a library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrQ-RapQ-ComA QS system to dynamically control the synthesis of MK-4 in B. subtilis 168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of MK-4 in shake flask from 120.1 ± 0.6 to 178.9 ± 2.8 mg/L, and reached 217 ± 4.1 mg/L in a 3-L bioreactor, which verified the effectiveness of this strategy. In summary, PhrQ-RapQ-ComA QS system can realize dynamic pathway regulation in B. subtilis 168, which can be stretched to a great deal of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Combinatorial engineering for improved menaquinone-4 biosynthesis in Bacillus subtilis.

Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is difficult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA, menG, and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P43 resulted in 8.1 ± 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 ± 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr, and ispD-ispF genes in MEP module with strong promoter P43 increased the titer of MK-4 to 78.1 ± 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaK1, mvaK2, mvaD, mvaS, and mvaA) resulted in 90.1 ± 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA, and menG in menaquinone pathway with strong promoter P43 increased the titer of MK-4 to 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future.

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis.

Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7 Synthesis in Bacillus subtilis.

Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

Enzyme Assembly for Compartmentalized Metabolic Flux Control.

Enzyme assembly by ligand binding or physically sequestrating enzymes, substrates, or metabolites into isolated compartments can bring key molecules closer to enhance the flux of a metabolic pathway. The emergence of enzyme assembly has provided both opportunities and challenges for metabolic engineering. At present, with the development of synthetic biology and systems biology, a variety of enzyme assembly strategies have been proposed, from the initial direct enzyme fusion to scaffold-free assembly, as well as artificial scaffolds, such as nucleic acid/protein scaffolds, and even some more complex physical compartments. These assembly strategies have been explored and applied to the synthesis of various important bio-based products, and have achieved different degrees of success. Despite some achievements, enzyme assembly, especially in vivo, still has many problems that have attracted significant attention from researchers. Here, we focus on some selected examples to review recent research on scaffold-free strategies, synthetic artificial scaffolds, and physical compartments for enzyme assembly or pathway sequestration, and we discuss their notable advances. In addition, the potential applications and challenges in the applications are highlighted.

Identification and Characterization of a Cellodextrin Transporter in Aspergillus niger.

Aspergillus niger produces a wide spectrum of extracellular polysaccharide hydrolases that hydrolyze cellulose into soluble glucose and cellodextrins. Transporters are essential for sugar uptake, yet it is not clear whether cellodextrin transporters exist in A. niger. Here, one cellulose inducible cellodextrin transporter CtA was identified in A. niger B2. It was found that CtA not only could transport cellobiose, but also cellotriose, cellotetraose, and cellopentaose. The yeast strain YPßG-CtA, expressing cellodextrin transporter CtA and an intracellular ß-glucosidase, grew on cellobiose with the cell growth rate of 0.0830 ± 0.0113 h-1 under aerobic condition. Furthermore, the engineered yeast could produce 1.1 g/L ethanol anaerobically on cellobiose in 2 days. The identification of CtA provides evidence that the cellodextrin uptake is a complementary strategy of cellulose utilization in A. niger, and the CtA could be a strong transporter candidate for constructing engineered cellodextrin-utilizing microorganisms.

Cell Membrane and Electron Transfer Engineering for Improved Synthesis of Menaquinone-7 in Bacillus subtilis.

The formation of biofilm facilitates the synthesis of valuable natural product menaquinone-7 (MK-7) in static culture of Bacillus subtilis, whereas the essential role and mechanism of biofilm in MK-7 synthesis have not been revealed. Herein, comparative transcriptomics show that the formation of biofilm affected MK-7 synthesis by changing the transcription levels of signal receptor (BSU02010), transmembrane transporter (BSU29340, BSU03070), and signal transduction (BSU02630). Moreover, we also found that oxalate decarboxylase OxdC has an important effect on electron generation and MK-7 synthesis, when the transcriptional level of NADH dehydrogenase decreases in static culture. Our results revealed that cell membrane and electron transfer are important factors in promoting MK-7 synthesis.

Enzyme assembly guided by SPFH-induced functional inclusion bodies for enhanced cascade biocatalysis.

Enzyme clustering into compact agglomerates could accelerate the processing of intermediates to enhance metabolic pathway flux. However, enzyme clustering is still a challenging task due to the lack of universal assembly strategy applicable to all enzymes. Therefore, we proposed an alternative enzyme assembly strategy based on functional inclusion bodies. First, functional inclusion bodies in cells were formed by the fusion expression of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain and enhanced green fluorescent protein, as observed visually and by transmission electron microscopy. The formation of SPFH-induced functional inclusion bodies enhanced intermolecular polymerization as revealed by further analysis combined with Förster resonance energy transfer and bimolecular fluorescent complimentary. Finally, the functional inclusion bodies significantly improved the enzymatic catalysis in living cells, as proven by the examples with whole-cell biocatalysis of phenyllactic acid by Escherichia coli, and the production of N-acetylglucosamine by Bacillus subtilis. Our findings suggest that SPFH-induced functional inclusion bodies can enhance the cascade reaction of enzymes, to serve as a potential universal strategy for the construction of efficient microbial cell factories.

Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives.

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

Engineering a Bifunctional Phr60-Rap60-Spo0A Quorum-Sensing Molecular Switch for Dynamic Fine-Tuning of Menaquinone-7 Synthesis in Bacillus subtilis.

Quorum sensing (QS)-based dynamic regulation has been widely used as basic tool for fine-tuning gene expression in response to cell density changes without adding expensive inducers. However, most reported QS systems primarily relied on down-regulation rather than up-regulation of gene expression, significantly limiting its potential as a molecular switch to control metabolic flux. To solve this challenge, we developed a bifunctional and modular Phr60-Rap60-Spo0A QS system, based on two native promoters, PabrB (down-regulation by Spo0A-P) and PspoiiA (up-regulation by Spo0A-P). We constructed a library of promoters with different capacities to implement down-regulation and up-regulation by changing the location, number, and sequences of the binding sites for Spo0A-P. The QS system can dynamically balance the relationship between efficient synthesis of the target product and cell growth. Finally, we validated the usefulness of this strategy by dynamic control of menaquinone-7 (MK-7) synthesis in Bacillus subtilis 168, a model Gram-positive bacterium, with the bifunctional Phr60-Rap60-Spo0A quorum sensing system. Our dynamic pathway regulation led to a 40-fold improvement of MK-7 production from 9 to 360 mg/L in shake flasks and 200 mg/L in 15-L bioreactor. Taken together, our bilayer QS system has been successfully integrated with biocatalytic functions to achieve dynamic pathway regulation in B. subtilis 168, which may be extended for use in other microbes to fine-tune gene expression and improve metabolites production.

Investigating the expression of F10 and G11 xylanases in Aspergillus niger A09 with qPCR.

There exist significant differences between the 2 main types of xylanases, family F10 and G11. A clear understanding of the expression pattern of microbial F10 and G11 under different culture conditions would facilitate better production and industrial application of xylanase. In this study, the fungal xylanase producer Aspergillus niger A09 was systematically investigated in terms of induced expression of xylanase F10 and G11. Results showed that carbon and nitrogen sources could influence xylanase F10 and G11 transcript abundance, with G11 more susceptible to changes in culture media composition. The most favorable carbon and nitrogen sources for high G11 and low F10 production by A. niger A09 were xylan (2%) and (NH4)2C2O4 (0.3%), respectively. Following cultivation at 33 °C for 60 h, the highest xylanase activity (1132 IU per gram of wet mycelia) was observed. On the basis of differential gene expression of F10 and G11, as well as their different properties, we deduced that the F10 protein initially targeted xylan and hydrolyzed it into fragments including xylose, after which xylose acted as the inducer of F10 and G11 gene expression. These speculations also accounted for our failure to identify conditions favoring the high production of F10 but a low production of G11.