Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.

While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.

Engineering sub-organelles of a diploid Saccharomyces cerevisiae to enhance the production of 7-dehydrocholesterol.

7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol.

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine.

l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E. coli, several genes associated with GSH and  l-cysteine degradation, as well as the branched metabolic flow, were deleted. Additionally, the GSH bifunctional synthase (GshFSA) and GSH ATP-binding cassette exporter (CydDC) were overexpressed. Moreover, an adenosine-based ATP regeneration system was first introduced into E. coli to enhance GSH biosynthesis without exogenous ATP additions. Through the optimization of whole-cell catalytic conditions, the engineered strain GSH17-FDC achieved an impressive GSH titer of 24.19 g/L only after 2 h reaction, with a nearly 100% (98.39%) conversion rate from the added  l-Cys. This work not only unveils a new platform for GSH production but also provides valuable insights for the production of other high-value metabolites that rely on ATP consumption.

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Advances in biotin biosynthesis and biotechnological production in microorganisms.

Biotin, also known as vitamin H or B7, acts as a crucial cofactor in the central metabolism processes of fatty acids, amino acids, and carbohydrates. Biotin has important applications in food additives, biomedicine, and other fields. While the ability to synthesize biotin de novo is confined to microorganisms and plants, humans and animals require substantial daily intake, primarily through dietary sources and intestinal microflora. Currently, chemical synthesis stands as the primary method for commercial biotin production, although microbial biotin production offers an environmentally sustainable alternative with promising prospects. This review presents a comprehensive overview of the pathways involved in de novo biotin synthesis in various species of microbes and insights into its regulatory and transport systems. Furthermore, diverse strategies are discussed to improve the biotin production here, including mutation breeding, rational metabolic engineering design, artificial genetic modification, and process optimization. The review also presents the potential strategies for addressing current challenges for industrial-scale bioproduction of biotin in the future. This review is very helpful for exploring efficient and sustainable strategies for large-scale biotin production.

Integration of a CRISPR Cas12a-assisted multicolor biosensor and a micropipette tip enables visible point-of-care testing of foodborne Vibrio vulnificus.

Foodborne pathogens cause numerous food safety problems, and as a virulent bacterium falling under this category, Vibrio vulnificus (V. vulnificus) poses a huge threat to public health. The conventional methods used for the detection of V. vulnificus, including culture-based and molecular detection methods, have a variety of drawbacks, including being time-consuming and labor-intensive, the requirement of large-scale equipment, and the lack of professional operators. This paper establishes a visible detection platform for V. vulnificus based on CRISPR/Cas12a, which is integrated with nucleic acid isothermal amplification and ß-galactosidase-catalyzed visible color reaction. The specific vvhA gene and a conservative segment in the 16S rDNA gene of the Vibrio genus were selected as the detection targets. By using spectrum analysis, this CRISPR detection platform achieved sensitive detection of V. vulnificus (1 CFU per reaction) with high specificity. Through the color transformation system, as low as 1 CFU per reaction of V. vulnificus in both bacterial solution and artificially contaminated seafood could be visibly observed with the naked eye. Furthermore, the consistency between our assay and the qPCR assay in the detection of V. vulnificus spiked seafood was confirmed. In general, this visible detection platform is user-friendly, accurate, portable, and equipment-free, and is expected to provide a powerful supplement in point-of-care testing of V. vulnificus and also holds good promise for future application in foodborne pathogen detection.

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per µL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

AIMS: To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis. MATERIALS AND RESULTS: This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously. CONCLUSIONS: This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens.

Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5'-phosphorylated and 5'-hydroxylated single-stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double-stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago-based DNA detection toolbox.

Efficient poly(ß-L-malic acid) production from cassava hydrolysate by cell recycle of Aureobasidium pullulans.

Poly(ß-L-malic acid) (PMLA) is a water-soluble, biodegradable, and biocompatible polymer with broad prospective applications and can be hydrolyzed to produce widely used acidulant L-malic acid. In order to meet an increasing demand of PMLA, we employed two effective cell-recycling strategies to produce PMLA from raw cassava hydrolysate by Aureobasidium pullulans ZD-3d. In fed-batch fermentation with raw cassava hydrolysate, 101.9 g/L PMLA was obtained with the productivity and yield of 0.77 g/L/h and 0.40 g/g, respectively. Further, three times of membrane filtration-based cell recycling fermentation was carried out, with a high productivity and yield of 1.04-1.64 g/L/h and 0.5-0.84 g/g achieved, respectively. While harnessing centrifugation-based cell recycling fermentation for five times, the productivity and yield approached 0.98-1.76 g/L/h and 0.78-0.86 g/g, respectively. To our knowledge, the processes showed the highest average PMLA productivity compared with others using low-cost biomass, which offered efficient and economical alternatives for PMLA production. KEY POINTS: • PMLA production from raw cassava hydrolysate by Aureobasidium pullulans was studied • High PMLA productivity and yield were obtained via two cell recycling strategies • The highest average PMLA productivity from low-cost biomass to date was achieved.

Microfluidic Ruler-Readout and CRISPR Cas12a-Responded Hydrogel-Integrated Paper-Based Analytical Devices (µReaCH-PAD) for Visible Quantitative Point-of-Care Testing of Invasive Fungi.

Invasive fungi (IF) have become a significant problem affecting human health. However, the culture-based assay of IF, known as the most commonly used clinical diagnostic method, suffers from time consumption, complicated operation, and the requirement of trained operators, which may cause the delay diagnosis of the disease. In this report, a microfluidic ruler-readout and CRISPR Cas12a-responded hydrogel-integrated paper-based analytical device (µReaCH-PAD) was established for visible and quantitative point-of-care testing of IF. Using the genus-conserved fragments of 18s rRNA as the detection target, this platform relied on a CRISPR Cas12a system for target recognition, a DNA hydrogel coupled with a cascade of enzymatic reactions for signal amplification and transduction, and paper-based microfluidic chips for visual quantitative readout by naked eyes. The 18s rRNA fragments of Candida or Aspergillus were employed as a model target and introduced with PAM sites for Cas12a-recognition during reverse transcription recombinase-aided amplification. Using µReaCH-PAD, as low as 10 CFU/mL Candida and Aspergillus were visually identified by unaided eyes. The calculated detection limits were 4.90 and 4.13 CFU/mL (in 1 mL samples), respectively. The quantitative detection results can be obtained in the range from 10 to 104 CFU/mL with reasonable specificity and accuracy compared with qRT-PCR. Furthermore, µReaCH-PAD can analyze complex biological samples by Candida, Aspergillus, and Cryptococcus detection systems and identify specific genera of different IF by naked eyes, indicating a good agreement with the culture-based assay and the advantages over G-testing and GM-testing systems. With the benefits of high sensitivity, selectivity, quantitative readout, low cost, and ease of operation, µReaCH-PAD is expected to provide a portable detection tool of IF in resource-limited settings by untrained personnel and technical support for early diagnosis.

Identification of novel metabolic engineering targets for S-adenosyl-L-methionine production in Saccharomyces cerevisiae via genome-scale engineering.

S-Adenosyl-L-methionine (SAM) is an important intracellular metabolite and widely used for treatment of various diseases. Although high level production of SAM had been achieved in yeast, novel metabolic engineering strategies are needed to further enhance SAM production for industrial applications. Here genome-scale engineering (GSE) was performed to identify new targets for SAM overproduction using the multi-functional genome-wide CRISPR (MAGIC) system, and the effects of these newly identified targets were further validated in industrial yeast strains. After 3 rounds of FACS screening and characterization, numerous novel targets for enhancing SAM production were identified. In addition, transcriptomic and metabolomic analyses were performed to investigate the molecular mechanisms for enhanced SAM accumulation. The best combination (upregulation of SNZ3, RFC4, and RPS18B) improved SAM productivity by 2.2-fold and 1.6-fold in laboratory and industrial yeast strains, respectively. Using GSE of laboratory yeast strains to guide industrial yeast strain engineering presents an effective approach to design microbial cell factories for industrial applications.

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae.

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

A CRISPR-Cas12a-derived biosensor enabling portable personal glucose meter readout for quantitative detection of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to achieve sensitive and specific detection of SARS-CoV-2. However, most methods are still largely dependent on professional instruments, highly trained operators, and centralized laboratories. These limitations gravely diminish their practicality and portability. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a based assay was developed for portable, rapid and sensitive of SARS-CoV-2. In this assay, samples were quickly pretreated and amplified by reverse transcription recombinase-aided amplification under mild conditions. Then, by combining the CRISPR Cas12a system and a glucose-producing reaction, the signal of the virus was converted to that of glucose, which can be quantitatively read by a personal glucose meter in a few seconds. Nucleocapsid protein gene was tested as a model target, and the sensitivity for quantitative detection was as low as 10 copies/µl, which basically meet the needs of clinical diagnosis. In addition, with the advantages of lower material cost, shorter detection time, and no requirement for professional instrument in comparison with quantitative reverse transcription-polymerase chain reaction, this assay is expected to provide a powerful technical support for the early diagnosis and intervention during epidemic prevention and control.

Functional expression of eukaryotic cytochrome P450s in yeast.

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms. Due to their key roles in secondary metabolism, degradation of xenobiotics, and carcinogenesis, there is a great demand to heterologously express and obtain a sufficient amount of active eukaryotic P450s. However, most eukaryotic P450s are endoplasmic reticulum-localized membrane proteins, which is the biggest challenge for functional expression to high levels. Furthermore, the functions of P450s require the cooperation of cytochrome P450 reductases for electron transfer. Great efforts have been devoted to the heterologous expression of eukaryotic P450s, and yeasts, particularly Saccharomyces cerevisiae are frequently considered as the first expression systems to be tested for this challenging purpose. This review discusses the strategies for improving the expression and activity of eukaryotic P450s in yeasts, followed by examples of P450s involved in biosynthetic pathway engineering.

Toxicity of soil antimony to earthworm Eisenia fetida (Savingy) before and after the aging process.

Information on soil antimony (Sb) toxicity to earthworm Eisenia fetida (Savingy) is limited. This ecotoxicology study was designed to quantify the soil Sb toxicity to earthworm E. fetida before and after aging process, establishing dose-effect relationship between Sb content and mortality. Results of the avoidance test and acute test showed that the values of net avoidance response, escape rate and mortality were generally decreased in aged treatment compared to that in fresh treatment, respectively from 93.33% to 66.67%, 36.67% to 13.33% and 100% to 53.33% (15 d) taking TL800 (treatment level of 800 mg/kg) for example, meanwhile the values of median lethal content (LC50) at 72 h, 7 d and 15 d were respectively increased from 355.27 mg/kg to 2324.55 mg/kg, 322.19 mg/kg and 1743.19 mg/kg and 282.74 mg/kg to 745.94 mg/kg, indicating that aging process could reduce the Sb acute toxicity to earthworm. According to a three-step sequential extraction procedure, the bioavailable Sb ranged from 24.45% to 43.24% and 16.97% to 27.70% in fresh treatment and aged treatment, respectively, and the mortality of earthworm for 24 h decreased with the decrease of the content of mild acid-soluble antimony (which decreased averagely from 23.09% to 14.00%), which was more suited to assess Sb toxicity. This is the first report that confirms the toxicity of soil Sb to earthworm E. fetida as well as the considering of aging process and speciation.

Multi-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin.

Biotin (Vitamin H or B7) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.

Efficient biotransformation of vitamin D3 to 25-hydroxyvitamin D3 by a newly isolated Bacillus cereus strain.

25-hydroxyvitamin D3 has attracted considerable attention due to its great medical value and huge market demand in animal husbandry. Microbial production of 25-hydroxyvitamin D3 has been recognized as an alternative superior to traditional chemical synthesis. In this study, a Gram-positive bacteria zju 4-2 (CCTCC M 2019385) was isolated from the soil using vitamin D3 as the sole carbon source and was identified as Bacillus cereus according to its physiological characteristics and 16S rRNA analysis, which also showed a relatively high capacity for 25-hydroxyvitamin D3 production. Through systematic optimization of different catalytic conditions, the optimal solvent system of vitamin D3, vitamin D3 addition time and concentration, temperature, and pH were shown to be propylene glycol/ethanol (v/v = 9:1), early stationary phase, 2 g/L, 37 °C, and pH 7.2, respectively. With these optimal conditions, 796 mg/L of 25-hydroxyvitamin D3 was achieved after 48 h bioconversion with zju 4-2 at the shake flask level. Finally, up to 830 mg/L 25-hydroxyvitamin D3 with a yield of 41.5% was obtained in a 5 L fermentation tank. Our developed biotransformation process with this newly isolated strain provides a platform to produce 25-hydroxyvitamin D3 efficiently at industrialization scale.

Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway.

5-Hydroxytryptophan (5-HTP) is the precursor of the neurotransmitter serotonin and has been used for the treatment of various diseases such as depression, insomnia, chronic headaches, and binge eating associated obesity. The production of 5-HTP had been achieved in our previous report, by the development of a recombinant strain containing two plasmids for biosynthesis of L-tryptophan (L-trp) and subsequent hydroxylation. In this study, the L-trp biosynthetic pathway was further integrated into the E. coli genome, and the promoter strength of 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first step of L-trp biosynthesis, was engineered to increase the production of L-trp. Hence, the 5-HTP production could be manipulated by the regulation of copy number of L-trp hydroxylation plasmid. Finally, the 5-HTP production was increased to 1.61 g/L in the shaking flasks, which was 24% improvement comparing to the original producing strain, while the content of residual L-trp was successfully reduced from 1.66 to 0.2 g/L, which is beneficial for the downstream separation and purification. Our work shall promote feasible progresses for the industrial production of 5-HTP.