De novo biosynthesis of betulinic acid in engineered Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupinane-type pentacyclic triterpenoid natural product derived from lupeol that has favorable anti-inflammatory and anti-tumor activities. Currently, BA is mainly produced via botanical extraction, which significantly limits its widespread use. In this study, we investigated the de novo synthesis of BA in Saccharomyces cerevisiae, and to facilitate the synthesis and storage of hydrophobic BA, we adopted a dual-engineering strategy involving peroxisomes and lipid droplets to construct the BA biosynthetic pathway. By expressing Betula platyphylla-derived lupeol C-28 oxidase (BPLO) and Arabidopsis-derived ATR1, we succeeded in developing a BA-producing strain and following multiple expression optimizations of the linker between BPLO and ATR1, the BA titer reached 77.53 mg/L in shake flasks and subsequently reached 205.74 mg/L via fed-batch fermentation in a 5-L bioreactor. In this study, we developed a feasible approach for the de novo synthesis of BA and its direct precursor lupeol in engineered S. cerevisiae.

Synergistic increase in coproporphyrin III biosynthesis by mitochondrial compartmentalization in engineered Saccharomyces cerevisiae.

Coproporphyrin III (CP III), a natural porphyrin derivative, has extensive applications in the biomedical and material industries. S. cerevisiae has previously been engineered to highly accumulate the CP III precursor 5-aminolevulinic acid (ALA) through the C4 pathway. In this study, a combination of cytoplasmic metabolic engineering and mitochondrial compartmentalization was used to enhance CP III production in S. cerevisiae. By integrating pathway genes into the chromosome, the CP III titer gradually increased to 32.5 ± 0.5 mg/L in shake flask cultivation. Nevertheless, increasing the copy number of pathway genes did not consistently enhance CP III synthesis. Hence, the partial synthesis pathway was compartmentalized in mitochondria to evaluate its effectiveness in increasing CP III production. Subsequently, by superimposing the mitochondrial compartmentalization strategy on cytoplasmic metabolic engineered strains, the CP III titer was increased to 64.3 ± 1.9 mg/L. Furthermore, augmenting antioxidant pathway genes to reduce reactive oxygen species (ROS) levels effectively improved the growth of engineered strains, resulting in a further increase in the CP III titer to 82.9 ± 1.4 mg/L. Fed-batch fermentations in a 5 L bioreactor achieved a titer of 402.8 ± 9.3 mg/L for CP III. This study provides a new perspective on engineered yeast for the microbial production of porphyrins.

Hypoxia-Inducible Factor-1α-Activated Protein Switch Based on Allosteric Self-Splicing Reduces Nonspecific Cytotoxicity of Pharmaceutical Drugs.

Protein-based therapeutic agents currently used for targeted tumor therapy exhibit limited penetrability, nonspecific toxicity, and a short circulation half-life. Although targeting cell surface receptors improves cancer selectivity, the receptors are also slightly expressed in normal cells; consequently, the nonspecific toxicity of recombinant protein-based therapeutic agents has not been eliminated. In this study, an allosteric-regulated protein switch was designed that achieved cytoplasmic reorganization of engineered immunotoxins in tumor cells via interactions between allosteric self-splicing elements and cancer markers. It can target the accumulated HIF-1α in hypoxic cancer cells and undergo allosteric activation, and the splicing products were present in hypoxic cancer cells but were absent in normoxic cells, selectively killing tumor cells and reducing nonspecific toxicity to normal cells. The engineered pro-protein provides a platform for targeted therapy of tumors while offering a novel universal strategy for combining the activation of therapeutic functions with specific cancer markers. The allosteric self-splicing element is a powerful tool that significantly reduces the nonspecific cytotoxicity of therapeutic proteins.

Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme.

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H2O2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.

Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol.

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (kcat/Km) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol.

One-Pot Two-Stage Biocatalytic Cascade to Produce l-Phosphinothricin by Two Enantioselective Complementary Aminotransferases at High Substrate Loading via a Deracemization Process.

The bioderacemization of racemic phosphinothricin (D, L-PPT) is a promising route for the synthesis of l-phosphinothricin (L-PPT). However, the low activity and tolerance of wild-type enzymes restrict their industrial applications. Two stereocomplementary aminotransferases with high activity and substrate tolerance were identified in a metagenomic library, and a one-pot, two-stage artificial cascade biocatalytic system was developed to produce L-PPT through kinetic resolution and asymmetric amination. We observed that 500 mM D, L-PPT (100 g/L) could be converted into L-PPT with 94% final conversion and >99.9% enantiomeric excess (e.e.) within 24 h, with only 0.02 eq amino acceptor pyruvate and 1.2 eq amino donor l-aspartate required. The process could be scaled up to 10 L under sufficient oxygen and stirring. The superior catalytic performance of this system provides an eco-friendly and sustainable approach to the industrial deracemization of D, L-PPT to L-PPT.

Engineering non-conventional yeast Rhodotorula toruloides for ergothioneine production.

BACKGROUND: Ergothioneine (EGT) is a distinctive sulfur-containing histidine derivative, which has been recognized as a high-value antioxidant and cytoprotectant, and has a wide range of applications in food, medical, and cosmetic fields. Currently, microbial fermentation is a promising method to produce EGT as its advantages of green environmental protection, mild fermentation condition, and low production cost. However, due to the low-efficiency biosynthetic process in numerous cell factories, it is still a challenge to realize the industrial biopreparation of EGT. The non-conventional yeast Rhodotorula toruloides is considered as a potential candidate for EGT production, thanks to its safety for animals and natural ability to synthesize EGT. Nevertheless, its synthesis efficiency of EGT deserves further improvement. RESULTS: In this study, out of five target wild-type R. toruloides strains, R. toruloides 2.1389 (RT1389) was found to accumulate the highest EGT production, which could reach 79.0 mg/L at the shake flask level on the 7th day. To achieve iterative genome editing in strain RT1389, CRISPR-assisted Cre recombination (CACR) method was established. Based on it, an EGT-overproducing strain RT1389-2 was constructed by integrating an additional copy of EGT biosynthetic core genes RtEGT1 and RtEGT2 into the genome, the EGT titer of which was 1.5-fold increase over RT1389. As the supply of S-adenosylmethionine was identified as a key factor determining EGT production in strain RT1389, subsequently, a series of gene modifications including S-adenosylmethionine rebalancing were integrated into the strain RT1389-2, and the resulting mutants were rapidly screened according to their EGT production titers with a high-throughput screening method based on ergothionase. As a result, an engineered strain named as RT1389-3 was selected with a production titer of 267.4 mg/L EGT after 168 h in a 50 mL modified fermentation medium. CONCLUSIONS: This study characterized the EGT production capacity of these engineered strains, and demonstrated that CACR and high-throughput screening method allowed rapid engineering of R. toruloides mutants with improved EGT production. Furthermore, this study provided an engineered RT1389-3 strain with remarkable EGT production performance, which had potential industrial application prospects.

Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

[Development and exploration of the national first-class undergraduate course in "Enzyme Engineering"].

The biotechnology industry is a strategic emerging industry in our country, holding a crucial position in the national economy. The training of innovative high-quality professionals carries immense significance. As the cornerstone course in biotechnology, "Enzyme Engineering" directly impacts the students' caliber and industry development. This course aims to address pertinent issues present in the current curriculum delivery, such as inadequately optimized content, excessive dependency on textbooks, and reliance on monotonous teaching methods. By adjusting the course outline, updating the case material repository, and engaging students' enthusiasm, we developed a three-dimensional approach to instruct. This approach included a blend of online and offline components, interactive teaching through the flipped classroom methodology, heuristic teaching using problem-based learning (PBL) mode, and topic teaching via case studies. We also improved the assessment mechanism to stimulate students' enthusiasm for learning nurture their innovation capabilities. Our objective was to foster high-quality professionals with a robust foundation and practical expertise. Through teaching exploration and practice, we have witnessed significant improvement in both teaching efficacy and students' engineering practice and innovation abilities.

Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol.

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency (kcat/KM) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the Tm value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.

CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Lactiplantibacillus plantarum X7022 Plays Roles on Aging Mice with Memory Impairment Induced by D-Galactose Through Restoring Neuronal Damage, Relieving Inflammation and Oxidative Stress.

In this study, Lactiplantibacillus plantarum X7022 was applied to ameliorate memory impairment of aging mice induced by D-galactose. The strain showed specific choloylglycine hydrolysis ability based on in vitro investigation. Morris water maze test showed L. plantarum X7022 administration improved learning ability and spatial memory of aging mice. The gavage of L. plantarum X7022 displayed a promising ability of relieving cerebral oxidative stress and hippocampal inflammatory condition according to the increased GSH level and SOD activity and decreased MDA level, as well as decreased TNF-α, IL-1ß, and IL-6 levels. The intervention with the strain could protect neuron by regulating cell apoptosis and AChE overexpression and inhibiting amyloid-ß deposition, as well as affect neuron functions by regulating CREB-BDNF signaling pathways and iNOS expression. Besides, the strain could improve fecal SCFA contents and increase the abundance of anti-inflammatory and antioxidant-related genera such as Lactobacillus, Akkermansia, and Adlercreutzia. These results suggest that L. plantarum X7022 could be a prospective therapeutic alternative for the improvement of memory impairment among the elderly.

Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement.

α-Amylase (EC 3.2.1.1) from Geobacillus stearothermophilus (generally recognized as safe) exhibited thermal inactivation, hampering its further application in starch-based industries. To address this, we performed structural analyses based on molecular dynamics targeting the flexible regions of α-amylase. Subsequently, we rationally designed a thermostable mutant, AmyS1, by introducing disulfide bonds to stabilize the flexible regions. AmyS1 showed excellent thermostability without any stability-activity trade-off, giving a 40-fold longer T1/2 (1359 min) at 90 °C. Thermostability mechanism analysis revealed that the introduction of disulfide bonds in AmyS1 refined weak spots and reconfigured the protein's force network. Moreover, AmyS1 exhibited improved pH compatibility and enhanced corn starch liquefaction at 100 °C with a 5.1-fold increased product concentration. Baking tests confirmed that AmyS1 enhanced bread quality and extended the shelf life. Therefore, mutant AmyS1 is a robust candidate for the starch-based industry.

Construction of RNA silencing system of Penicillium brevicompactum and genetic manipulation of the regulator pbpcz in mycophenolic acid production.

Penicillium brevicompactum is a critical industrial strain for the production of mycophenolic acid (MPA). However, the genetic background of Penicillium brevicompactum is unclear, and there are few tools available for genetic manipulation. To investigate its gene function, we first verified the feasibility of a pair of citrate synthase promoter (Pcit) and terminator (Tcit) from P. brevicompactum by constructing a fluorescent expression cassette. Based on this, an RNAi vector was designed and constructed with reverse promoters. This study focused on the functional investigation of the pbpcz gene in P. brevicompactum, a regulator belonging to the Zn(II)2Cys6 family. RNAi was used to silence the pbpcz gene, providing a valuable tool for genetic studies in P. brevicompactum. After seven days, we observed differences in the number of spores between different phenotypes strains of pbpcz gene. Compared to the wild-type strain (WT), the spore yield of the pbpcz gene silencing mutant (M2) was only 51.4 %, while that of the pbpcz gene overexpressed mutant (SE4) was increased by 50 %. Expression levels of the three genes (brlA, abaA, and wetA) comprising conidia's central regulatory pathway were significantly reduced in the pbpcz gene silencing mutant, while fluorescence localization showed that PbPCZ protein was mainly distributed in spores. The results indicated that the pbpcz gene is critical for conidia and asexual development of P. brevicompactum. In addition, overexpressing the pbpcz gene resulted in a 30.3 % increase in MPA production compared to the wild type, with a final yield of 3.57 g/L. These results provide evidence that PbPCZ acts as a positive regulator in P. brevicompactum, controlling MPA production and regulating conidia and asexual development.

Identification, Characterization, and Computer-Aided Rational Design of a Novel Thermophilic Esterase from Geobacillus subterraneus, and Application in the Synthesis of Cinnamyl Acetate.

Investigation of a novel thermophilic esterase gene from Geobacillus subterraneus DSMZ 13552 indicated a high amino acid sequence similarity of 25.9% to a reported esterase from Geobacillus sp. A strategy that integrated computer-aided rational design tools was developed to select mutation sites. Six mutants were selected from four criteria based on the simulated saturation mutation (including 19 amino acid residues) results. Of these, the mutants Q78Y and G119A were found to retain 87% and 27% activity after incubation at 70 °C for 20 min, compared with the 19% activity for the wild type. Subsequently, a double-point mutant (Q78Y/G119A) was obtained and identified with optimal temperature increase from 65 to 70 °C and a 41.51% decrease in Km. The obtained T1/2 values of 42.2 min (70 °C) and 16.9 min (75 °C) for Q78Y/G119A showed increases of 340% and 412% compared with that in the wild type. Q78Y/G119A was then employed as a biocatalyst to synthesize cinnamyl acetate, for which the conversion rate reached 99.40% with 0.3 M cinnamyl alcohol at 60 °C. The results validated the enhanced enzymatic properties of the mutant and indicated better prospects for industrial application as compared to that in the wild type. This study reported a method by which an enzyme could evolve to achieve enhanced thermostability, thereby increasing its potential for industrial applications, which could also be expanded to other esterases.

Engineering the metabolism and morphology of the filamentous fungus Trichoderma reesei for efficient L-malic acid production.

L-malic acid (MA) is a vital platform chemical with huge market demand because of its broad industrial applications. A cell factory for MA production was engineered by strengthening the intrinsic pathway without inserting foreign genes into Trichoderma reesei. The native MA transporter gene in the T. reesei genome was characterized (trmae1), and its overexpression significantly improved MA production, which increased from 2 to 56.24 g/L. Native pyruvate carboxylase, malate dehydrogenase, malic enzyme, and glucose transporter were overexpressed further to improve the titer and yield of MA production. Fungal morphology was adapted to produce MA in the fermenter by deleting gul1. A titer of 235.8 g/L MA was produced from the final engineered strain in a 5-L fermenter with a yield of 1.48 mol of MA per mol of glucose and productivity of 1.23 g/L/h. This study provides novel insights for understanding and remodeling the MA synthesis pathway.

Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons.

BACKGROUND: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. RESULTS: The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. CONCLUSIONS: The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.

Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: • Cryptic plasmids of EcN were designed to express exogenous genes • Insertion sites with different expression intensities in cryptic plasmids were provided • Target products were stably produced by engineering cryptic plasmids.