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[Display omitted] • High levels of Dac secretion were realized by signal peptide engineering. • The efficient expression of Dac were realized by RBS sequence optimization. • The stable and efficient expression of Dac was achieved through genomic integration. • B. subtilis C6 was constructed by CRISPR/Cpf1 system to facilitate gene editing. • The extracellular Dac activity(6357.38 U/mL) was the highest level reported so far. Diacetylchitobiose deacetylase (Dac) from Pyrococcus horikoshii can realize the one-step production of glucosamine (GlcN). The efficient expression and secretion of Dac play a central role in the green production of GlcN. In this study, Bacillus subtilis WB600 was used as the expression host. Firstly, we screened 12 signal peptides, among which signal peptide NprB had the strongest ability of guiding Dac secretion. Further optimization of the functional region showed that the extracellular Dac activity of NprB mutant was increased to 3682.2 U/mL. Next, the extracellular Dac activity was increased to 4807.6 U/mL by RBS sequence optimization. Then we got a new recombinant B. subtilis C6 for plasmid-free expression of Dac by integrating comK gene and silencing bpr , nprB , aprE , mpr and nprE genes. Finally, the extracellular Dac activity of genome-integrating strain reached 6357.38 U/mL, which was the highest level reported so far. [ABSTRACT FROM AUTHOR]

• After mutation screening of selected promoters, promoter P A11 displayed highest expression intensity. • PhrQ-RapQ-ComA QS system was constructed to dynamically control the synthesis of MK-4 in B. subtilis 168. • Our dynamic adjustment method increased the yield of MK-4 to 217 ± 4.1 mg/L in a 3-L bioreactor. 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 P A11 , 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. [ABSTRACT FROM AUTHOR]

• Overexpression of menA , menG , and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module resulted in 8.1 ± 0.2 mg/L of MK-4. • By knocking out hepT gene, the titer of MK-4 in the resultant strain BY03 was increased by 3.8-fold. • The MK-4 titer reached 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor, respectively. 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 P 43 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 P 43 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 P 43 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. [ABSTRACT FROM AUTHOR]

7-Dehydrocholesterol (7-DHC), a direct precursor of vitamin D3, has attracted increasing attention in microbial fermentation recently. In this study, 7-DHC biosynthesis in diploid Saccharomyces cerevisiae with robust ergosterol production was achieved by heterologous 24-dehydrocholesterol reductase expression, generating 44.1 mg/L 7-DHC, whereas the titer of ergosterol decreased by 40.5%. The ergosterol biosynthetic pathway was completely blocked by knocking out ERG6 and ERG5 , affording a 4.2-fold increase in the 7-DHC titer. Subsequently, the facilitation of the mevalonate and the postsqualene pathways accompanied by elimination of transcriptional repressors enhanced 7-DHC synthesis, and the 7-DHC titer reached 738.5 mg/L in a shake flask. Further validation in a 50 L fermenter demonstrated that the 7-DHC titer reached 3.80 g/L within just 24 h, with productivity reaching 158.3 mg/L/h, setting a new benchmark as the highest reported to date. This study paves the way toward a large-scale and cost-effective manufacture of 7-DHC.

Given the extensive heterogeneity and variability, understanding cellular functions and regulatory mechanisms through the analysis of multi-omics datasets becomes extremely challenging. Here, a comprehensive modeling framework of multi-omics machine learning and metabolic network models are proposed that covers various cellular biological processes across multiple scales. This model on an extensive normalized compendium of Bacillus subtilis is validated, which encompasses gene expression data from environmental perturbations, transcriptional regulation, signal transduction, protein translation, and growth measurements. Comparison with high-throughput experimental data shows that EM_iBsu1209-ME, constructed on this basis, can accurately predict the expression of 605 genes and the synthesis of 23 metabolites under different conditions. This study paves the way for the construction of comprehensive biological databases and high-performance multi-omics metabolic models to achieve accurate predictive analysis in exploring complex mechanisms of cell genotypes and phenotypes., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Long non-coding RNAs were commonly viewed as non-coding elements. However, they are increasingly recognized for their ability to be translated into proteins, thereby playing a significant role in various cellular processes and diseases. With developments in biotechnology and computational algorithms, a range of novel approaches are being applied to investigate the translation of long non-coding RNA (lncRNAs). Herein, we developed the LncPepAtlas database (http://www.cnitbiotool.net/LncPepAtlas/), which aims to compile multiple evidences for the translation of lncRNAs and annotations for the upstream regulation of lncRNAs across various species. LncPepAtlas integrated compelling evidence from nine distinct sources for the translation of lncRNAs. These include a dataset comprising 2631 publicly available Ribo-seq samples from nine species, which has been collected and analysed. LncPepAtlas offers extensive annotation for lncRNA upstream regulation and expression profiles across various cancers, tissues or cell lines at transcriptional and translational levels. Importantly, it enables novel antigen predictions for lncRNA-encoded peptides. By identifying numerous peptide candidates that could potentially bind to major histocompatibility complex class I and II molecules, this work may provide new insights into cancer immunotherapy. The function of peptides were inferred by aligning them with experimentally detected proteins. LncPepAtlas aims to become a convenient resource for exploring translatable lncRNAs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lipid droplets (LDs) are specialized organelles that store neutral lipids to reduce the negative effects of lipotoxicity on cells. However, many neutral lipids are precursors for the synthesis of sterols and complex terpenoids, and this sequestration often greatly limits the efficient biosynthesis of sterols and complex terpenoids. In this study, taking 7-dehydrocholesterol (7-DHC) synthesis in Saccharomyces cerevisiae as an example, we revealed the blocking mechanism of LD sequestration on the efficient synthesis of metabolic products and found that LDs can sequester a significant amount of squalene, the precursor of 7-DHC, effectively preventing it from being directed toward the post-squalene pathway. Based on this, a post-squalene pathway was reconstructed on LDs, which resulted in a 28.7% increase in the 7-DHC titer, reaching 684.1 mg/L, whereas the squalene titer was reduced by approximately 97%. Subsequently, the triacylglycerol degradation pathway was weakened to release the storage space in LDs, and the esterification pathway was concurrently strengthened to guide 7-DHC storage within LDs, which further increased 7-DHC production, reaching 792.9 mg/L. Finally, by reducing the NADH/NAD + ratio to alleviate the redox imbalance, the 7-DHC titer reached 867.6 mg/L in shake flask and 5.1 g/L in a 3-L bioreactor, which is the highest reported titer to date. In summary, this study provides new insights into the important role of LDs in sterol synthesis and offers a novel strategy for constructing cell factories for the efficient synthesis of sterol compounds., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction., (© 2024 Wiley Periodicals LLC.)

There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities., (© 2024. The Author(s).)

Ovalbumin (OVA) is a high-quality protein for humans. Modifying microorganisms to produce proteins offers a solution to potential food protein shortages. In this study, OVA was expressed in Saccharomyces cerevisiae . Initially, screening signal peptides led to extracellular OVA reaching 3.4 mg/L using the INU1 signal peptide. Coexpressing Kar2 and PDI increased OVA production to 5.1 mg/L. Optimizing the expression levels of regulators OPI1, INO2, and INO4 expanded the endoplasmic reticulum membrane, raising yield to 5.5 mg/L. Combining both strategies increased OVA production to 6.2 mg/L, 82% higher than control. This strategy also enhanced secretion of other proteins. Finally, fed-batch fermentation in a 3-L bioreactor significantly boosted OVA production to 116.3 mg/L. This study provides insights for the heterologous synthesis of other high-quality proteins for future food applications.

Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties. This review categorizes current continuous evolution into three distinct classes: non-targeted chromosomal, targeted chromosomal, and extra-chromosomal hypermutation approaches. It also compares various continuous evolution strategies based on different principles, providing a reference for selecting suitable methods for specific evolutionary goals. Furthermore, this review discusses the two primary limitations for further widespread application of in vivo continuous evolution, which are lack of general applicability and insufficient mutagenic capability. We envision that developing generally applicable mutagenic components and methods to enhance mutation rates for in vivo continuous evolution are promising future directions for wide range applications of continuous evolution., (© 2024 Wiley-VCH GmbH.)

In recent years, genetic circuit-based regulation of metabolic flux in microbial cell factories has received significant attention. In this review, we describe a pipeline for the design and construction of genetic circuits for metabolic flux optimization. In particular, we summarize the recent advances in computationally assisted prediction of critical metabolic nodes and genetic circuit design automation. Further, we introduce strategies for constructing high-performance genetic circuits. We also summarize the latest applications of genetic circuits in the dynamic regulation of metabolism and high-throughput screening. Finally, we discuss the challenges and prospects associated with the design and construction of sophisticated genetic circuits. Through this review, we aim to provide a theoretical basis for designing and constructing high-performance genetic circuits to optimize metabolic flux., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P 21 , P 43 , and P veg ) and three inducible promoters (P grac100 , P xylA , and P tet * ) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 μg/L, was achieved when regulated by the P tet * promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SP yycP resulted in increased HLF expression to 1 099.87 μg/L, with 498.68 μg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 μg/L. This study successfully demonstrated the secretory expression of HLF in B . subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B . subtilis cell factories for lactoprotein synthesis.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli . α-1,3-Fucosyltransferase ( futM 2 ) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (k cat /K m ) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol -1 reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in k cat-GlcNAc6P /K m-GlcNAc6P and a 59% decrease in k cat-Glc6P /K m-Glc6P is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L -1 with a yield of 0.597 g (g glucose) -1 in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD + and NADPH/NADP + increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 μg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 μg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA , encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 μg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L -1 in shake flasks using the OVA expression cassette containing a tac promoter (P tac ) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (P P5 combined with P tac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L -1 , respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L -1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmid-based multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the " BacAmp ", a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis , which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N -acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis ., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors.)

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 P AOX1 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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Production of food-related products using microorganisms in an environmentally friendly manner is a crucial solution to global food safety and environmental pollution issues. Traditional microbial modification methods rely on artificial selection or natural mutations, which require time for repeated screening and reproduction, leading to unstable results. Therefore, it is imperative to develop rapid, efficient, and precise microbial modification technologies. This review summarizes recent advances in the construction of gene editing and metabolic regulation toolkits based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) systems and their applications in reconstructing food microorganism metabolic networks. The development and application of gene editing toolkits from single-site gene editing to multi-site and genome-scale gene editing was also introduced. Moreover, it presented a detailed introduction to CRISPR interference, CRISPR activation, and logic circuit toolkits for metabolic network regulation. Moreover, the current challenges and future prospects for developing CRISPR genetic toolkits were also discussed., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023. Published by Elsevier Inc.)

The industrial yeast Komagataella phaffii is a highly effective platform for heterologous protein production, owing to its high protein expression and secretion capacity. Heterologous genes and proteins are involved in multiple processes, including transcription, translation, protein folding, modification, transportation, and degradation; however, engineering these proteins and genes is challenging due to inefficient genome editing techniques. We employed Pseudomonas aeruginosa phage single-stranded DNA-annealing protein (SSAP) PapRecT and P. aeruginosa single-stranded DNA-binding protein (SSB) PaSSB to introduce SSAP-SSB-based homology recombination, which facilitated K. phaffii CRISPR-based genome engineering. Specifically, a host-independent method was developed by expressing sgRNA with PapRecT-PaSSB in a single plasmid, with which only a 50 bp short homologous arm (HA) reached a 100% positive rate for CRISPR-based gene insertion, reaching 18 colony-forming units (CFU) per μg of donor DNA. Single deletion using 1000 bp HA attained 100%, reaching 68 CFUs per μg of donor DNA. Using this efficient CRISPR-based genome editing tool, we integrated three genes ( INO4 , GAL4-like , and PAB1 ) at three different loci for overexpression to realize the collaborative regulation of human-lactalbumin (α-LA) production. Specifically, we strengthened phospholipid biosynthesis to facilitate endoplasmic reticulum membrane formation and enhanced recombinant protein transcription and translation by overexpressing transcription and translation factors. The final production of α-LA in the 3 L fermentation reached 113.4 mg L -1 , two times higher than that of the strain without multiple site gene editing, which is the highest reported titer in K. phaffii . The CRISPR-based genome editing method developed in this study is suitable for the synergistic multiple-site engineering of protein and biochemical biosynthesis pathways to improve the biomanufacturing efficiency.

Background: Saccharomyces cerevisiae has been used in the biosynthesis of acid products such as organic acids owing to its acid tolerance. Improving the acid tolerance of S. cerevisiae is beneficial for expanding its application range. Our previous study isolated the TAMC strain that was tolerant to a pH 2.3 through adaptive laboratory evolution; however, its mechanism underlying tolerance to low pH environment remains unclear., Results: In this study, through visual observation and order analysis of plasma membrane and membrane microdomains, we revealed that the membrane microdomains of TAMC strain play an indispensable role in acid tolerance. Transcriptomic analysis showed an increase in the expression of genes related to key components of membrane microdomains in TAMC strain. Furthermore, an obvious reduction was observed in the acid tolerance of the strain with sterol C-24 methyltransferase encoding gene ERG6 knockout for inhibiting membrane microdomain formation. Finally, colocalization analysis of H + -ATPase PMA1 and plasma membrane protein PMP1 showed that disruption of membrane microdomains could inhibit the formation of the H + -ATPase complex., Conclusions: Membrane microdomains could provide a platform for forming H + -ATPase complexes to facilitate intracellular H + homeostasis, and thereby improve cell acid resistance. This study proposed a novel acid tolerance mechanism, providing a new direction for the rational engineering of acid-tolerant strains., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The increasing global population and protein demand cause global challenges for food supply. Fueled by significant developments in synthetic biology, microbial cell factories are constructed for the bioproduction of milk proteins, providing a promising approach for scalable and cost-effective production of alternative proteins. This review focused on the synthetic biology-based microbial cell factory construction for milk protein bioproduction. The composition, content, and functions of major milk proteins were first summarized, especially for caseins, α-lactalbumin, and β-lactoglobulin. An economic analysis was performed to determine whether cell factory-based milk protein production is economically viable for industrial production. Cell factory-based milk protein production is proved to be economically viable for industrial production. However, there still exist some challenges for cell factory-based milk protein biomanufacturing and application, including the inefficient production of milk proteins, insufficient investigation of protein functional property, and insufficient food safety evaluation. Constructing new high-efficiency genetic regulatory elements and genome editing tools, coexpression/overexpression of chaperone genes, and engineering protein secretion pathways and establishing a cost-effective protein purification method are possible ways to improve the production efficiency. Milk protein biomanufacturing is one of the promising approaches to acquiring alternative proteins in the future, which is of great importance for supporting cellular agriculture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Limonene is a volatile monoterpene compound that is widely used in food additives, pharmaceutical products, fragrances, and toiletries. We herein attempted to perform efficient biosynthesis of limonene in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, we conducted de novo synthesis of limonene in S. cerevisiae and achieved a titer of 46.96 mg/L. Next, by dynamic inhibition of the competitive bypass of key metabolic branches regulated by ERG20 and optimization of the copy number of tLimS , a greater proportion of the metabolic flow was directed toward limonene synthesis, achieving a titer of 640.87 mg/L. Subsequently, we enhanced the acetyl-CoA and NADPH supply, which increased the limonene titer to 1097.43 mg/L. Then, we reconstructed the limonene synthesis pathway in the mitochondria. Dual regulation of cytoplasmic and mitochondrial metabolism further increased the limonene titer to 1586 mg/L. After optimization of the process of fed-batch fermentation, the limonene titer reached 2.63 g/L, the highest ever reported in S. cerevisiae .

l-Histidine is an essential proteinogenic amino acid in food with extensive applications in the pharmaceutical field. Herein, we constructed a Corynebacterium glutamicum recombinant strain for efficient biosynthesis of l-histidine. First, to alleviate the l-histidine feedback inhibition, the ATP phosphoribosyltransferase mutant HisG T235P-Y56M was constructed based on molecular docking and high-throughput screening, resulting in the accumulation of 0.83 g/L of l-histidine. Next, we overexpressed rate-limiting enzymes including HisG T235P-Y56M and PRPP synthetase and knocked out the pgi gene in the competing pathway, which increased the l-histidine production to 1.21 g/L. Furthermore, the energy status was optimized by decreasing the reactive oxygen species level and enhancing the supply of adenosine triphosphate, reaching a titer of 3.10 g/L in a shake flask. The final recombinant strain produced 5.07 g/L of l-histidine in a 3 L bioreactor, without the addition of antibiotics and chemical inducers. Overall, this study developed an efficient cell factory for l-histidine biosynthesis by combinatorial protein engineering and metabolic engineering.

The metabolic burden caused by terpenoid accumulation limits the development of highly efficient microbial cell factories, which can be circumvented using exporter-mediated product secretion. Although our previous study showed that the pleiotropic drug resistance exporter (PDR11) mediates the export of rubusoside in Saccharomyces cerevisiae , the underlying mechanism is still unclear. Herein, we used GROMACS software to simulate PDR11-mediated rubusoside recruitment and found six residues (D116, D167, Y168, P521, R663, and L1146) on PDR11 that are critical for this process. We also explored the exportation potential of PDR11 for 39 terpenoids by calculating their binding affinity using batch molecular docking. Then, we verified the accuracy of the predicted results by conducting experiments with squalene, lycopene, and β-carotene as examples. We found that PDR11 can efficiently secrete terpenoids with binding affinities lower than -9.0 kcal/mol. Combining the computer-based prediction and experimental verification, we proved that binding affinity is a reliable parameter to screen exporter substrates and might potentially enable rapid screening of exporters for natural products in microbial cell factories.

Optimizing the metabolic pathways of microbial cell factories is essential for establishing viable biotechnological production processes. However, due to the limited understanding of the complex setup of cellular machinery, building efficient microbial cell factories remains tedious and time-consuming. Machine learning (ML), a powerful tool capable of identifying patterns within large datasets, has been used to analyze biological datasets generated using various high-throughput technologies to build data-driven models for complex bioprocesses. In addition, ML can also be integrated with Design-Build-Test-Learn to accelerate development. This review focuses on recent ML applications in genome-scale metabolic model construction, multistep pathway optimization, rate-limiting enzyme engineering, and gene regulatory element designing. In addition, we have discussed some limitations of these methods as well as potential solutions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter -1 in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Bacillus subtilis is a microbial cell factory widely used to produce recombinant proteins, but the expression of heterologous proteins is often severely hampered. This study constructed a genetic toolkit for improving the secretory efficiency of heterologous proteins in Bacillus subtilis. First, the protease-deficient hosts were reconstructed. Then, two endogenous constitutive promoters, P hag and P spovG , were screened. Next, a method called systemic combinatorial optimization of ribosome binding site (RBS) equipped with signal peptide (SCORES) was designed for optimizing the secretion and translation of the heterologous protein. Finally, Serratia marcescens nonspecific endonuclease (SMNE), which causes cell death by degrading nucleic acids, was expressed. The enzyme activity in the shake flask reached 7.5 × 10 6 U/L, which was 7.5-times that of the control RBS and signal peptide combination (RS0). This study not only expanded on the synthetic biology toolbox in B. subtilis but also provided strategies to create a prokaryotic protein expression system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Menthol, a high-value commodity monoterpenoid chemical, holds an important market share commercially because of its distinct functions. The menthol on the market mainly originates from plant extraction, which is facing challenges such as the seasonal fluctuations and long growth cycle of plants. Therefore, this study attempted to realize the de novo synthesis of menthol through microbial fermentation. First, through heterologous expression and subcellular localization observation, a synthetic route from glucose to (-)-menthol was successfully designed and constructed in Saccharomyces cerevisiae . Then, the mevalonate (MVA) pathway was enhanced, and the expression of farnesyl diphosphate synthase ( ERG20 ) was dynamically regulated to improve the synthesis of D-limonene, a key precursor of (-)-menthol. Shake flask fermentation results showed that the D-limonene titer of the recombinant strain reached 459.59 mg/L. Next, the synthesis pathway from D-limonene to (-)-menthol was strengthened, and the fermentation medium was optimized. The (-)-menthol titer of 6.28 mg/L was obtained, implying that the de novo synthesis of menthol was successfully realized for the first time. This study provides a good foundation for the synthesis of menthol through microbial fermentation., Competing Interests: The authors declare no conflicts of interest.

Microscopy imaging of living cells is becoming a pivotal, noninvasive, and highly specific tool in metabolic engineering to visualize molecular dynamics in industrial microorganisms. This review describes the different microscopy methods, from fluorescence to super resolution, with application in microbial bioengineering. Firstly, the role and importance of microscopy imaging is analyzed in the context of strain design. Then, the advantages and disadvantages of different microscopy technologies are discussed, including confocal laser scanning microscopy (CLSM), spatial light interference microscopy (SLIM), and super-resolution microscopy, followed by their applications in synthetic biology. Finally, the future perspectives of live-cell imaging and their potential to transform microbial systems are analyzed. This review provides theoretical guidance and highlights the importance of microscopy in understanding and engineering microbial metabolism., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

High-sugar diet causes health problems, many of which can be addressed with the use of sugar substitutes. Rubusoside and rebaudiosides are interesting molecules, considered the next generation of sugar substitutes due to their low-calorie, superior sweetness and organoleptic properties. However, their low abundance in nature makes the traditional plant extraction process neither economical nor environmental-friendly. Here we engineer baker's yeast Saccharomyces cerevisiae as a chassis for the de novo production of rubusoside and rebaudiosides. In this process, we identify multiple issues that limit the production, including rate-liming steps, product stress on cellular fitness and unbalanced metabolic networks. We carry out a systematic engineering strategy to solve these issues, which produces rubusoside and rebaudiosides at titers of 1368.6 mg/L and 132.7 mg/L, respectively. The rubusoside chassis strain here constructed paves the way towards a sustainable, large-scale fermentation-based manufacturing of diverse rebaudiosides., (© 2022. The Author(s).)

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.

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 OD 600 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., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The anchoring of metabolic pathway enzymes to spatial scaffolds can significantly improve their reaction efficiency. Here, we successfully constructed a multi-enzyme complex assembly system able to enhance bioproduction in bacteria by using the endogenous spatial scaffolds─functional membrane microdomains (FMMs). First, using VA-TIRFM and SPT analysis, we reveal that FMMs possess high temporal and spatial stability at the plasma membrane and can be used as endogenous spatial scaffolds to organize enzyme pathways. Then, taking the synthesis of N-acetylglucosamine (GlcNAc) in Bacillus subtilis as a proof-of-concept demonstration, we found that anchoring of various enzymes required for GlcNAc synthesis onto FMMs to obtain the FMMs-multi-enzyme complex system resulted in a significant increase in GlcNAc titer and an effectively alleviate in cell lysis at the later stage of fermentation compared to that in control strains expressing the related enzymes in the cytoplasm. Combining with metabolic model and kinetics analysis, the existence of a constructed substrate channel that maximizes the reaction efficiency is verified. In summary, we propose a novel metabolic pathway assembly model which allowed improved titers and compartmentalized flux control with high spatial resolution in bacterial metabolism., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

This paper presents the design, fabrication, and characterization of a middle-infrared (MIR) linear variable optical filter (LVOF) and thermopile detectors that will be used in a miniaturized mixed gas detector for CH 4 /CO 2 /CO measurement. The LVOF was designed as a tapered-cavity Fabry-Pérot optical filter, which can transform the MIR continuous spectrum into multiple narrow band-pass spectra with peak wavelength in linear variation. Multi-layer dielectric structures were used to fabricate the Bragg reflectors on the both sides of tapered cavity as well as the antireflective film combined with the function of out-of-band rejection. The uncooled thermopile detectors were designed and fabricated as a multiple-thermocouple suspension structure using micro-electro-mechanical system technology. Experimentally, the LVOF exhibits a mean full-width-at-half-maximum of 400 nm and mean peak transmittance of 70% at the wavelength range of 2.3~5 μm. The thermopile detectors exhibit a responsivity of 146 μV/°C at the condition of room temperature. It is demonstrated that the detectors can achieve the quantification and identification of CH 4 /CO 2 /CO mixed gas.

High frequency surface acoustic wave (SAW) technology offers many opportunities for aerospace applications in passive wireless sensing and communication. This paper presents the design, simulation, fabrication, and test of an L -band SAW resonator based on 128° Y-X LiNbO 3 substrate. The design parameters of SAW resonator were optimized by the finite element (FEM) method and the coupling-of-mode (COM) theory. Electron-beam lithography (EBL) technology was used to fabricate the submicron-scale of interdigital transducers (IDTs) and grating reflectors. The effects of some key EBL processes (e.g., the use of electron beam resist, the choice of metal deposition methods, the charge-accumulation effect, and the proximity-effect) on the fabrication precision of SAW devices were discussed. Experimentally, the LiNbO 3 -based SAW resonators fabricated using improved EBL technology exhibits a Rayleigh wave resonance peaks at 1.55 GHz with return loss about -12dB, and quality factor Q is 517. Based on this SAW resonator, the temperature and strain sensing tests were performed, respectively. The experimental results exhibit a well linear dependence of temperature/strain on frequency-shift, with a temperature sensitivity of 125.4 kHz/C and a strain sensitivity of -831 Hz/με, respectively., Competing Interests: The authors declare no conflict of interest.

One of the primary goals of microbial metabolic engineering is to achieve high titer, yield and productivity (TYP) of engineered strains. This TYP index requires optimized carbon flux toward desired molecule with minimal by-product formation. De novo redesign of central carbon and redox metabolism holds great promise to alleviate pathway bottleneck and improve carbon and energy utilization efficiency. The engineered strain, with the overexpression or deletion of multiple genes, typically can't meet the TYP index, due to overflow of central carbon and redox metabolism that compromise the final yield, despite a high titer or productivity might be achieved. To solve this challenge, we reprogramed the central carbon and redox metabolism of Bacillus subtilis and achieved high TYP production of N-acetylglucosamine. Specifically, a "push-pull-promote" approach efficiently reduced the overflown acetyl-CoA flux and eliminated byproduct formation. Four synthetic NAD(P)-independent metabolic routes were introduced to rewire the redox metabolism to minimize energy loss. Implementation of these genetic strategies led us to obtain a B. subtilis strain with superior TYP index. GlcNAc titer in shake flask was increased from 6.6 g L -1 to 24.5 g L -1 , the yield was improved from 0.115 to 0.468 g GlcNAc g -1 glucose, and the productivity was increased from 0.274 to 0.437 g L -1 h -1 . These titer and yield are the highest levels ever reported and, the yield reached 98% of the theoretical pathway yield (0.478 g g -1 glucose). The synthetic redesign of carbon metabolism and redox metabolism represent a novel and general metabolic engineering strategy to improve the performance of microbial cell factories., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The compound 2,5-furandicarboxylic acid (FDCA), an important bio-based monomer for the production of various polymers, can be obtained from 5-hydroxymethylfurfural (HMF). However, efficient production of FDCA from HMF via biocatalysis has not been well studied. In this study, we report the identification of key genes that are involved in FDCA synthesis and then the engineering of Raoultella ornithinolytica BF60 for biocatalytic oxidation of HMF to FDCA using its resting cells. Specifically, previously unknown candidate genes, adhP3 and alkR , which were responsible for the reduction of HMF to the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol), were identified by transcriptomic analysis. Combinatorial deletion of these two genes resulted in 85.7% reduction in HMF alcohol formation and 23.7% improvement in FDCA production (242.0 mM). Subsequently, an aldehyde dehydrogenase, AldH, which was responsible for the oxidation of the intermediate 5-formyl-2-furoic acid (FFA) to FDCA, was identified and characterized. Finally, FDCA production was further improved by overexpressing AldH, resulting in a 96.2% yield of 264.7 mM FDCA. Importantly, the identification of these key genes not only contributes to our understanding of the FDCA synthesis pathway in R. ornithinolytica BF60 but also allows for improved FDCA production efficiency. Moreover, this work is likely to provide a valuable reference for producing other furanic chemicals.

Phospholipase D has great commercial value due to its transphosphatidylation products that can be used in the food and medicine industries. In order to construct a strain for use in the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region. We found that using the signal peptide amyE results in the highest extracellular PLD activity (11.3 U/ml) and in a PLD expression level 5.27-fold higher than when the endogenous signal peptide is used. Furthermore, the strain harboring the recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by 69.03% (19.1 U/ml). We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activity by about 26.7% (24.2 U/ml). In addition, we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%. The final product, phosphatidylserine (PS), was successfully detected, with transphosphatidylation selectivity at 74.6%. This is similar to the values for the original producer.