Search

Nicotinamide mononucleotide (NMN) serves as a precursor for NAD + synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the de novo NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered Escherichia coli to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield. Subsequently, we progressively enhanced the de novo NMN biosynthesis route to further augment NMN production. We screened and identified the crucial role of MazG in catalyzing the enzymatic cleavage of NAD + to NMN. And the de novo NMN biosynthesis route was optimized and integrated with the NadV-mediated NMN biosynthetic pathways, leading to an intracellular concentration of 844.10 ± 17.40 μM NMN. Furthermore, the introduction of two transporters enhanced the uptake of NAM and the excretion of NMN, resulting in NMN production of 1293.73 ± 61.38 μM. Finally, by engineering an E. coli strain with optimized PRPP synthetase, we achieved the highest NMN production, reaching 3067.98 ± 27.25 μM after 24 h of fermentation at the shake flask level. In addition to constructing an efficient E. coli cell factory for NMN production, our findings provide new insights into understanding the NAD + salvage pathway and its role in energy metabolism within E. coli .

Background: Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative., Results: This study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the -35 region and into the spacer region between the -10 and -35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 μM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 μM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants., Conclusions: The methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production., (© 2024. The Author(s).)

Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR-based chromosome-doubling technique is designed to first construct an artificial diploid Escherichia coli cell. The stable single-cell diploid E. coli is isolated by both maximal dilution plating and flow cytometry, and confirmed with quantitative PCR, fluorescent in situ hybridization, and third-generation genome sequencing. The diploid E. coli has a greatly reduced growth rate and elongated cells at 4-5 µm. It is robust against radiation, and the survival rate after exposure to UV increased 40-fold relative to WT. As a novel life form, the artificial diploid E. coli is an ideal substrate for research fundamental questions in life science concerning polyploidy. And this technique may be applied to other bacteria., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Currently, the biological production of L-malic acid (L-MA) is mainly based on the fermentation of filamentous fungi at near-neutral pH, but this process requires large amounts of neutralizing agents, resulting in the generation of waste salts when free acid is obtained in the downstream process, and the environmental hazards associated with the waste salts limit the practical application of this process. To produce L-MA in a more environmentally friendly way, we metabolically engineered the acid-tolerant yeast Pichia kudriavzevii and achieved efficient production of L-MA through low pH fermentation. First, an initial L-MA-producing strain that relies on the reductive tricarboxylic acid (rTCA) pathway was constructed. Subsequently, the L-MA titer and yield were further increased by fine-tuning the flux between the pyruvate and oxaloacetate nodes. In addition, we found that the insufficient supply of NADH for cytoplasmic malate dehydrogenase (MDH) hindered the L-MA production at low pH, which was resolved by overexpressing the soluble pyridine nucleotide transhydrogenase SthA from E. coli. Transcriptomic and metabolomic data showed that overexpression of EcSthA contributed to the activation of the pentose phosphate pathway and provided additional reducing power for MDH by converting NADPH to NADH. Furthermore, overexpression of EcSthA was found to help reduce the accumulation of the by-product pyruvate but had no effect on the accumulation of succinate. In microaerobic batch fermentation in a 5-L fermenter, the best strain, MA009-10-URA3 produced 199.4 g/L L-MA with a yield of 0.94 g/g glucose (1.27 mol/mol), with a productivity of 1.86 g/L/h. The final pH of the fermentation broth was approximately 3.10, meaning that the amount of neutralizer used was reduced by more than 50% compared to the common fermentation processes using filamentous fungi. To our knowledge, this is the first report of the efficient bioproduction of L-MA at low pH and represents the highest yield of L-MA in yeasts reported to date., Competing Interests: Declaration of competing interest This work has been included in patent applications by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains., Results: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli 0.10/4.54 and E. coli 2.28/2.36 . The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli 0.10/4.54 was quite stable, the two chromosomes of E. coli 2.28/2.36 population recombined into a new chromosome (Chr.4.64M Mut ), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated., Conclusion: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future., (© 2022. The Author(s).)

Background: The bifunctional enzyme β-carotene hydroxylase (CrtZ) catalyzes the hydroxylation of carotenoid β-ionone rings at the 3, 3' position regardless of the presence of keto group at 4, 4' position, which is an important step in the synthesis of astaxanthin. The level and substrate preference of CrtZ may have great effect on the amount of astaxanthin and the accumulation of intermediates., Results: In this study, the substrate preference of PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from Pantoea Agglomerans were certified and were combined utilization for increase astaxanthin production. Firstly, PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from P. Agglomerans were expressed in platform strains CAR032 (β-carotene producing strain) and Can004 (canthaxanthin producing strain) separately to identify their substrate preference for carotenoids with keto groups at 4,4' position or not. The results showed that PCcrtZ led to a lower zeaxanthin yield in CAR032 compared to that of PAcrtZ. On the contrary, higher astaxanthin production was obtained in Can004 by PCcrtZ than that of PAcrtZ. This demonstrated that PCCrtZ has higher canthaxanthin to astaxanthin conversion ability than PACrtZ, while PACrtZ prefer using β-carotene as substrate. Finally, Ast010, which has two copies of PAcrtZ and one copy of PCcrtZ produced 1.82 g/L of astaxanthin after 70 h of fed-batch fermentation., Conclusions: Combined utilization of crtZ genes, which have β-carotene and canthaxanthin substrate preference respectively, can greatly enhance the production of astaxanthin and increase the ratio of astaxanthin among total carotenoids., (© 2022. The Author(s).)

L-alanine is extensively used in chemical, food, and medicine industries. Industrial production of L-alanine has been mainly based on the enzymatic process using petroleum-based L-aspartic acid as the substrate. L-alanine production from renewable biomass using microbial fermentation process is an alternative route. Many microorganisms can naturally produce L-alanine using aminotransferase or L-alanine dehydrogenase. However, production of L-alanine using the native strains has been limited due to their low yields and productivities. In this review, metabolic engineering of microorganisms for L-alanine production was summarized. Among them, the Escherichia coli strains developed by Dr. Lonnie Ingram's group which can produce L-alanine with anaerobic fermentation process had several advantages, especially having high L-alanine yield, and it was the first one that realized commercialization. L-alanine is also the first amino acid that could be industrially produced by anaerobic fermentation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Glycolate is a bulk chemical with wide applications in the textile, food processing, and pharmaceutical industries. Glycolate can be produced from glucose via the glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. However, two problems limit the productivity and yield of glycolate when using glucose as the sole carbon source. The first is a cofactor imbalance in the production of glycolate from glucose via the glycolysis pathway, since NADPH is required for glycolate production, while glycolysis generates NADH. To rectify this imbalance, the NADP + -dependent glyceraldehyde 3-phosphate dehydrogenase GapC from Clostridium acetobutylicum was introduced to generate NADPH instead of NADH in the oxidation of glyceraldehyde 3-phosphate during glycolysis. The soluble transhydrogenase SthA was further eliminated to conserve NADPH by blocking its conversion into NADH. The second problem is an unfavorable carbon flux distribution between the tricarboxylic acid cycle and the glyoxylate shunt. To solve this problem, isocitrate dehydrogenase (ICDH) was eliminated to increase the carbon flux of glyoxylate and thereby improve the glycolate titer. After engineering through the integration of gapC, combined with the inactivation of ICDH, SthA, and by-product pathways, as well as the upregulation of the two key enzymes isocitrate lyase (encoding by aceA), and glyoxylate reductase (encoding by ycdW), the glycolate titer increased to 5.3 g/L with a yield of 1.89 mol/mol glucose. Moreover, an optimized fed-batch fermentation reached a titer of 41 g/L with a yield of 1.87 mol/mol glucose after 60 h., (© 2021 Wiley Periodicals LLC.)

As an important model industrial microorganism, Escherichia coli has been widely used in pharmaceutical, chemical industry and agriculture. In the past 30 years, a variety of new strategies and techniques, including artificial intelligence, gene editing, metabolic pathway assembly, and dynamic regulation have been used to design, construct, and optimize E. coli cell factories, which remarkably improved the efficiency for biotechnological production of chemicals. In this review, three key aspects for constructing E. coli cell factories, including pathway design, pathway assembly and regulation, and optimization of global cellular performance, are summarized. The technologies that have played important roles in metabolic engineering of E. coli, as well as their future applications, are discussed.

Astaxanthin has great potential commercial value in the feed, cosmetics, and nutraceutical industries due to its strong antioxidant capacity. In this study, the Escherichia coli strain CAR026 with completely balanced metabolic flow was selected as the starting strain for the production of astaxanthin. The expression of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ), which catalyze the conversion of β-carotene to astaxanthin, was coordinated, and a bottleneck was eliminated by increasing the copy number of crtY in CAR026. The resulting strain Ast007 produced 21.36 mg/L and 4.6 mg/g DCW of astaxanthin in shake flasks. In addition, the molecular chaperone genes groES - groEL were regulated to further improve the astaxanthin yield. The best strain Gro-46 produced 26 mg/L astaxanthin with a yield of 6.17 mg/g DCW in shake flasks and 1.18 g/L astaxanthin after 60 h of fermentation under fed-batch conditions. To the best of our knowledge, this is the highest astaxanthin obtained using engineered E. coli to date.

Background: Deactivated Cas9 (dCas9) led to significant improvement of CRISPR/Cas9-based techniques because it can be fused with a variety of functional groups to form diverse molecular devices, which can manipulate or modify target DNA cassettes. One important metabolic engineering strategy is to localize the enzymes in proximity of their substrates for improved catalytic efficiency. In this work, we developed a novel molecular device to manipulate the cellular location of specific DNA cassettes either on plasmids or on the chromosome, by fusing location tags to dCas9 (Cas9-Lag), and applied the technique for synthetic biology applications. Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane compartment., Results: Carotenoids like β-carotene serve as common intermediates for the synthesis of derivative compounds, which are hydrophobic and usually accumulate in the membrane components. To improve the functional expression of membrane-bound enzymes and localize them in proximity to the substrates, Cas9-Lag was used to pull plasmids or chromosomal DNA expressing carotenoid enzymes onto the cell membrane. For this purpose, dCas9 was fused to the E. coli membrane docking tag GlpF, and gRNA was designed to direct this fusion protein to the DNA expression cassettes. With Cas9-Lag, the zeaxanthin and astaxanthin titer increased by 29.0% and 26.7% respectively. Due to experimental limitations, the electron microscopy images of cells expressing Cas9-Lag vaguely indicated that GlpF-Cas9 might have pulled the target DNA cassettes in close proximity to membrane. Similarly, protein mass spectrometry analysis of membrane proteins suggested an increased expression of carotenoid-converting enzymes in the membrane components., Conclusion: This work therefore provides a novel molecular device, Cas9-Lag, which was proved to increase zeaxanthin and astaxanthin production and might be used to manipulate DNA cassette location.

Glycolate is an important α-hydroxy acid with a wide range of industrial applications. The current industrial production of glycolate mainly depends on chemical synthesis, but biochemical production from renewable resources using engineered microorganisms is increasingly viewed as an attractive alternative. Crude glycerol is an abundant byproduct of biodiesel production and a widely investigated potential sustainable feedstock. Here, we constructed a novel biosynthetic pathway for the production of glycolate from glycerol in Escherichia coli . The pathway starts from the oxidation of glycerol to d-glycerate by alditol oxidase, followed by sequential enzymatic dehydrogenation and decarboxylation as well as reduction reactions. We screened and characterized the catalytic activity of candidate enzymes, and a variant of alditol oxidase from Streptomyces coelicolor A3(2), 2-hydroxyglutarate-pyruvate transhydrogenase from Saccharomyces cerevisiae , α-ketoisovalerate decarboxylase from Lactococcus lactis , and aldehyde dehydrogenase from Escherichia coli were selected and assembled to create an artificial operon for the biosynthetic production of glycolate from glycerol. We also characterized the native strong constitutive promoter P lpp from E. coli and compared it with the P T7 promoter, which was employed to express the artificial operon on the plasmid pSC105-ADKA. To redirect glycerol flux toward glycolate synthesis, we deleted key genes of the native glycerol assimilation pathways and other branches of native E. coli metabolism, and we introduced a second plasmid expressing Dld3 to reduce the accumulation of the intermediate d-glycerate. Finally, the engineered strain TZ-108 harboring pSC105-ADKA and pACYC184-P lpp -Dld3 produced 0.64 g/L glycolate in shake flasks, which was increased to 4.74 g/L in fed-batch fermentation. This study provides an alternative pathway for glycolate synthesis and demonstrates the potential for producing other commodity chemicals by redesigning glycerol metabolism.

Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO 2 . In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21's defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21's to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.

The introduction of the mevalonate pathway (MVA pathway) in recombinant Escherichia coli can improve the synthesis of terpenoids. But the imbalance expression of MVA pathway genes and accumulation of intermediates inhibit cell growth and terpenoids production. In this study, each gene of MVA pathway and key genes of lycopene synthesis pathway were cloned in plasmid to express in the recombinant E. coli LYC103 with optimizing the expression of the key genes of the 2-methyl-D-erythritol-4-phosphate pathway (MEP pathway), chromosome recombinant MVA pathway and the lycopene synthesis pathway. The results showed that the overexpression of ispA, crtE, mvaK1, idi and mvaD genes did not affect the cell growth, while lycopene production increased by 13.5%, 16.5%, 17.95%, 33.7% and 61.1% respectively, indicating that these genes may be the rate-limiting steps for the synthesis of lycopene. mvaK1, mvaK2, mvaD of MVA pathway were the rate-limiting steps and were in an operon. The mvaK1, mvaK2, mvaD operon was regulated by mRS (mRNA stabilizing region) library in front of mvaK1, obtaining strain LYC104. Lycopene yield of LYC104 was doubled and cell growth was increased by 32% compared with the control strain LYC103. CRISPR-cas9 technology was used to integrate idi into chromosome at lacZ site to obtain LYC105 strain. Cell growth of LYC105 was increased by 147% and lycopene yield was increased by 2.28 times compared with that of LYC103. In this study, each gene of lycopene synthesis pathway was expressed in plasmid to certify the rate-limiting gene based on the complete MVA pathway on the chromosome. Then the rate-limiting gene was integrated in chromosome with homologous recombination to release the rate-limiting, which providing a new strategy for the construction of high-yield strains for metabolic engineering.

A balanced and optimized metabolic pathway is the basis for efficient production of a target metabolite. Traditional strategies mostly involve the manipulation of promoters or ribosome-binding sites, which can encompass long sequences and can be complex to operate. In this work, we found that by changing only the three nucleotides of the initiation codons, expression libraries of reporter proteins RFP, GFP, and lacZ with a large dynamic range and evenly distributed expression levels could be established in Escherichia coli (E. coli). Thus, a novel strategy that uses combinatorial modulation of initial codons (CMIC) was developed for metabolic pathway optimization and applied to the three genes crtZ, crtY, and crtI of the zeaxanthin synthesis pathway in E. coli. The initial codons of these genes were changed to random nucleotides NNN, and the gene cassettes were assembled into vectors via an optimized strategy based on type II restriction enzymes. With minimal labor time, a combinatorial library was obtained containing strains with various zeaxanthin production levels, including a strain with a titer of 6.33 mg/L and specific production value of 1.24 mg/g DCW-a striking 10-fold improvement over the starting strain. The results demonstrated that CMIC was a feasible technique for conveniently optimizing metabolic pathways. To our best knowledge, this is the first metabolic engineering strategy that relies on manipulating the initiation codons for pathway optimization in E. coli., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Metabolic engineering of microorganisms for production of succinate from glycerol remains challenging. It was thought that energy supply is a severe problem for anaerobic fermentation of glycerol to produce succinate. In this study, an energy-conserving glycerol utilization pathway was recruited to improve anaerobic succinate production in Escherichia coli. ATP dependent dihydroxyacetone kinase (DhaK) from Klebsiella was used to replace the native phosphoenolpyruvate (PEP) dependent dihydroxyacetone kinase (DhaKLM) of E. coli so that 2 NADH, instead of 1 NADH and 1 menaquinol, can be produced from glycerol to PEP. Besides consumption of 1 NADH and 1 menaquinol for converting PEP to succinate, NADH dehydrogenase I transfers electrons from 1 NADH to 1 menaquinone and pumps 4 protons. Proton-motive force are generated as the additional energy to support cell growth and succinate export. After using this energy-conserving glycerol utilization pathway, succinate titer, productivity and intercellular ATP content increased 282%, 63% and 338% respectively. The best strain YY-GS004 produced 483 mM succinate in 96 h with a yield of 0.92 mol mol -1 glycerol under anaerobic conditions. The specific succinate productivity was 0.47 g g -1 (DCW) h -1 , which was the highest one up to date for succinate production from glycerol. This study demonstrated that the energy-conserving glycerol utilization pathway GldA-DhaK can solve the energy problem during anaerobic fermentation of glycerol to produce succinate., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Unlike eukaryotes, prokaryotes are less proficient in homologous recombination (HR) and non-homologous end-joining (NHEJ). All existing genomic editing methods for Escherichia coli (E. coli) rely on exogenous HR or NHEJ systems to repair DNA double-strand breaks (DSBs). Although an E. coli native end-joining (ENEJ) system has been reported, its potential in genetic engineering has not yet been explored. Here, we present a CRISPR-Cas9-assisted native end-joining editing and show that ENEJ-dependent DNA repair can be used to conduct rapid and efficient deletion of chromosome fragments up to 83 kb or gene inactivation. Moreover, the positive rate and editing efficiency are independent of high-efficiency competent cells. The method requires neither exogenous DNA repair systems nor introduced editing template. The Cas9-sgRNA complex is the only foreign element in this method. This study is the first successful engineering effort to utilize ENEJ mechanism in genomic editing and provides an effective strategy for genetic engineering in bacteria that are inefficient in HR and NHEJ.

Background: Crocin is a carotenoid-derived natural product found in the stigma of Crocus spp., which has great potential in medicine, food and cosmetics. In recent years, microbial production of crocin has drawn increasing attention, but there were no reports of successful implementation. Escherichia coli has been engineered to produce various carotenoids, including lycopene, β-carotene and astaxanthin. Therefore, we intended to construct E. coli cell factories for crocin biosynthesis., Results: In this study, a heterologous crocetin and crocin synthesis pathway was first constructed in E. coli. Firstly, the three different zeaxanthin-cleaving dioxygenases CsZCD, CsCCD2 from Crocus sativus, and CaCCD2 from Crocus ancyrensis, as well as the glycosyltransferases UGT94E5 and UGT75L6 from Gardenia jasminoides, were introduced into zeaxanthin-producing E. coli cells. The results showed that CsCCD2 catalyzed the synthesis of crocetin dialdehyde. Next, the aldehyde dehydrogenases ALD3, ALD6 and ALD9 from Crocus sativus and ALD8 from Neurospora crassa were tested for crocetin dialdehyde oxidation, and we were able to produce 4.42 mg/L crocetin using strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8). Glycosyltransferases from diverse sources were screened by in vitro enzyme activity assays. The results showed that crocin and its various derivatives could be obtained using the glycosyltransferases YjiC, YdhE and YojK from Bacillus subtilis, and the corresponding genes were introduced into the previously constructed crocetin-producing strain. Finally, crocin-5 was detected among the fermentation products of strain YL4(pCsCCD2-UGT94E5-UGT75L6,pTrc-ALD8,pET28a-YjiC-YdhE-YojK) using HPLC and LC-ESI-MS., Conclusions: A heterologous crocin synthesis pathway was constructed in vitro, using glycosyltransferases from the Bacillus subtilis instead of the original plant glycosyltransferases, and a crocetin and crocin-5 producing E. coli cell factory was obtained. This research provides a foundation for the large-scale production of crocetin and crocin in E. coli cell factories.

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E. coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted β-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted β-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted β-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of β-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a β-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted β-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.

Isoprenoids are all derived from two five-carbon building blocks called isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized either by the mevalonate (MVA) pathway or 2-C-methyld-D-erythritol-4-phosphate (MEP) pathway. In this study, the MVA pathway genes were integrated into the chromosome of LYC101, in which the expression of key genes in the MEP synthesis pathway and lycopene synthesis pathway were optimized by artificial regulatory parts, to further improve the production of isoprenoids in Escherichia coli. The plasmids pALV23 and pALV145 were screened from a plasmid library that constructed by using the RBS library to link the genes of the MVA pathway, which greatly increased the production of β-carotene. The effects of plasmids pALV23 and pALV145 on the lycopene production in low and high lycopene production strain, LYC001 and LYC101, were compared, respectively. The production of lycopene was increased by plasmids pALV23 and pALV145 in both strains. In high lycopene production strain LYC101, pALV23 produced more lycopene than pALV145. Then, the MVA gene together of promoter of pALV23 was integrated into the chromosome of LYC101 at poxB site using method of homologous recombination helped by CRISPR-Cas9 system, resulted in genetically stable strain, LYC102. The yield of lycopene of LYC102 was 40.9 mg/g DCW, 1.19-folds higher than that of LYC101, and 20% more than that of LYC101 with pALV23. Simultaneous expression of MVA pathway and MEP pathway in recombinant E. coli can effectively increase the yield of terpenoids. In this study, a plasmid-free, genetically stable, high-yielding lycopene strain was constructed, which could be used for industrialization. Also, the platform strain can be used for the synthesis of other terpenoids.

A highly sensitive whole cell based electrochemical biosensor was developed for catechol detection in this study. The carE gene of Sphingobium yanoikuyae XLDN2-5 encoding catechol 2,3-dioxygenase (C23O), a key enzyme in the biodegradation of aromatic compound, was cloned and over-expressed in Escherichia coli BL21 (E. coli BL21). Compared to Sphingobium yanoikuyae XLDN2-5, the recombinant E. coli BL21 over-expressed C23O exhibited higher catalytic activity towards catechol. Moreover, the whole cells provided a better environment for C23O to maintain its catalytic activity and stability compared with crude enzyme. The distinctive features of the recombinant E. coli BL21 over-expressed C23O made it an ideal bio-recognition element for the fabrication of a microbial biosensor. Additionally, nanoporous gold (NPG) with unique properties of structure and function was selected as a support to immobilized the recombinant E. coli BL21 over-expressed C23O. Based on the synergistic effect of C23O and NPG, the E. coli BL21-C23O/NPG/GCE bioelectrode showed a good linear response for catechol detection ranging from 1 μM to 500 μM with a high sensitivity of 332.24 μA mM -1 cm -2 and a low detection limit of 0.24 μM. Besides, the E. coli BL21-C23O/NPG/GCE bioelectrode exhibited strong anti-interference and good stability. For the detection of catechol in wastewater samples, the concentrations detected by the E. coli BL21-C23O/NPG/GCE bioelectrode were in good agreement with the standard concentrations that added in the wastewater samples, which make the E. coli BL21-C23O/NPG/GCE bioelectrode an ideal tool for reliable catechol detection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Electrochemical energy is a key factor of biosynthesis, and is necessary for the reduction or assimilation of substrates such as CO 2 . Previous microbial electrosynthesis (MES) research mainly utilized naturally electroactive microbes to generate non-specific products., Results: In this research, an electroactive succinate-producing cell factory was engineered in E. coli T110(pMtrABC, pFccA-CymA) by expressing mtrABC, fccA and cymA from Shewanella oneidensis MR-1, which can utilize electricity to reduce fumarate. The electroactive T110 strain was further improved by incorporating a carbon concentration mechanism (CCM). This strain was fermented in an MES system with neutral red as the electron carrier and supplemented with HCO 3 + , which produced a succinate yield of 1.10 mol/mol glucose-a 1.6-fold improvement over the parent strain T110., Conclusions: The strain T110(pMtrABC, pFccA-CymA, pBTCA) is to our best knowledge the first electroactive microbial cell factory engineered to directly utilize electricity for the production of a specific product. Due to the versatility of the E. coli platform, this pioneering research opens the possibility of engineering various other cell factories to utilize electricity for bioproduction.

Genome editing for site-specific chromosome modification is one of the most significant techniques in biological research. While conventional techniques usually deal with one genomic locus at a time, multiple genomic targets are often required to be modified to develop microbial cell factories. Thus, it is necessary to develop techniques for simultaneous editing of multiple loci. In this work, the authors develop a CRISPR/Cas9 assisted multiplex genome editing (CMGE) technique in Escherichia coli. With this editing method, all functional parts are assembled into replicable plasmids, and stringent inducible expression systems are used to control Cas9 gene expression, which is to decouple transformation from editing process to increase editing efficiency. A modular assembly strategy is designed to enable construction of the complex multi-gRNA plasmid. With this technique, two and three loci are able to be modified with 100% and 88.3% efficiencies, while four loci can be edited with more than 30%, which are the best results reported. Although developed in model organism, the strategy of CMGE can be adapted to other prokaryotic cells. This is a well designed and illustrated technique with no special requirement, can be used by any biological lab easily., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The MEP pathway genes were modulated to investigate whether there were new rate-limiting steps and toxic intermediates in this pathway. Activating IspG led to significant decrease of cell growth and β-carotene production. It was found that ispG overexpression led to accumulation of intermediate HMBPP, which seriously interfered with synthesis machinery of nucleotide and protein in Escherichia coli. Activation of the downstream enzyme IspH could solve HMBPP accumulation problem and eliminate the negative effects of ispG overexpression. In addition, intermediate MECPP accumulated in the starting strain, while balanced activation of IspG and IspH could push the carbon flux away from MECPP and led to 73% and 77% increase of β-carotene and lycopene titer respectively. Our work for the first time identified HMBPP to be a cytotoxic intermediate in MEP pathway and demonstrated that balanced activation of IspG and IspH could eliminate accumulation of HMBPP and MECPP and improve isoprenoids production., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli. In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome. To implement it, two plasmids were employed to target Cas9 to regulatory sequences of pathway genes, and a donor DNA plasmid library was constructed containing a regulator pool to modulate the expression of these genes. A modularized plasmid construction strategy was used to enable the assembly of a complex donor DNA plasmid library. After genome editing using this technique, a combinatorial library was obtained with variably expressed pathway genes. As a demonstration, the CFPO technique was applied to the xylose metabolic pathway genes in E. coli to improve xylose utilization. Three transcriptional units containing a total of four genes were modulated simultaneously with 70% efficiency, and improved strains were selected from the resulting combinatorial library by growth enrichment. The best strain, HQ304, displayed a 3-fold increase of the xylose-utilization rate. Finally, the xylose-utilization pathway of HQ304 was analyzed enzymologically to determine the optimal combination of enzyme activities., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Biologically fixation of CO 2 has great potential as a significant carbon source for biosynthesis, which is also a major way to reduce CO 2 accumulation in atmosphere. Phosphoenolpyruvate (PEP) carboxylation is the key step of anaerobic succinate production in Escherichia coli. In this reaction, one mole CO 2 is assimilated with PEP to form oxaloacetate by PEP carboxykinase (PCK). The preferred substrate of PCK is CO 2 , which is very limited in cytoplasm. In this study, the carbon concentration mechanism (CCM) of cyanobacteria was introduced into Escherichia coli to enhance the intracellular inorganic carbon concentration for improving carboxylation velocity. Overexpression of the bicarbonate transporter (BT) or carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 led to a 22 or 35% increase in succinate titer at 36 h, respectively. The carboxylation rate of PCK increased from 2.46 to 3.92 µmol min -1  mg -1 protein by overexpression of the CA gene. In addition, co-overexpression of BT and CA genes had a synergetic effect, leading to a 44% increase in succinate titer at 36 h. This work is the first attempt to increase carbon fixation involved in microbial biosynthesis by engineering a biological CO 2 delivery system, which provides new direction and strategies for improving industrial fermentations based on biological CO 2 assimilation pathways., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carotenoids are a class of terpenes of commercial interest that exert important biological functions. While various strategies have been applied to engineer β-carotene production in microbial cell factories, no work has been done to study and improve the storage of hydrophobic terpene products inside the heterologous host cells. Although the membrane is thought to be the cell compartment that accumulates hydrophobic terpenes such as β-carotene, direct evidence is still lacking. In this work, we engineered the membrane of Escherichia coli in both its morphological and biosynthetic aspects, as a means to study and improve its storage capacity for β-carotene. Engineering the membrane morphology by overexpressing membrane-bending proteins resulted in a 28% increase of β-carotene specific producton value, while engineering the membrane synthesis pathway led to a 43% increase. Moreover, the combination of these two strategies had a synergistic effect, which caused a 2.9-fold increase of β-carotene specific production value (from 6.7 to 19.6mg/g DCW). Inward membrane stacks were observed in electron microscopy images of the engineered E. coli cells, which indicated that morphological changes were associated with the increased β-carotene storage capacity. Finally, membrane separation and analysis confirmed that the increased β-carotene was mainly accumulated within the cell membrane. This membrane engineering strategy was also applied to the β-carotene hyperproducing strain CAR025, which led to a 39% increase of the already high β-carotene specific production value (from 31.8 to 44.2mg/g DCW in shake flasks), resulting in one of the highest reported specific production values under comparable culture conditions. The membrane engineering strategy developed in this work opens up a new direction for engineering and improving microbial terpene producers. It is quite possible that a wide range of strains used to produce hydrophobic compounds can be further improved using this novel engineering strategy., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application., Results: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose., Conclusions: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Improvement in the osmotolerance of Escherichia coli is essential for the production of high titers of various bioproducts. In this work, a cusS mutation that was identified in the previously constructed high-succinate-producing E. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cells from Ag(I) and Cu(I) toxicity. Changing cusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of the cusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and a 40% increase in succinate titer. When the medium was supplemented with 30 g/liter disodium succinate, the cusS mutation led to a 120% increase in cell mass and a 492% increase in succinate titer. Introducing the cusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/liter disodium succinate. The cusS mutation increased the expression of cusCFBA , and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA may alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that can chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance of E. coli under anaerobic conditions. IMPORTANCE In this work, an activating Cus copper efflux system was found to increase the osmotolerance of E. coli In addition, new osmoprotectants were identified. Supplementation with methionine or cysteine led to an increase in osmotolerance of E. coli under anaerobic conditions. These new strategies for improving osmotolerance will be useful for improving the production of chemicals in industrial bioprocesses., (Copyright © 2017 American Society for Microbiology.)

Glycerol is a byproduct during biodiesel production. It is an important feedstock for fermentation due to its low price and high reduced status. Multiple genes of the glycerol utilization pathway were modulated in a previously engineered high β-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was firstly deleted. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts, M1-37, M1-46 and M1-93, respectively. β-carotene titer reached 64.82 mg/L after modulating glpD with M1-46, which was 4.86 times higher than that of CAR015, and glycerol consumption rate also increased 100%. Modulating tpiA led to a little increase of β-carotene titer, whereas modulating glpFK led to a little decrease of β-carotene titer. This demonstrated that GlpD was a rate-limiting step in glycerol utilization pathway. Q-PCR of glpF, glpK, glpD and tpiA results showed that decrease the transcription level of glpF, glpK, glpD, or decrease the transcription level of tpiA could increase the cell growth and β-carotene production, probably for the decrease of methylglyoxal toxicity. Modulating glpD and tpiA genes in combination resulted in the best strain Gly003, which produced 72.45 mg/L β-carotene with a yield of 18.65 mg/g dry cell weight. The titer was 5.23 and yield 1.99 times of that of the parent strain CAR015. Our work suggested that appropriate activation of glpD and tpiA genes in glycerol utilization pathway could effectively improve β-carotene production. This strategy can be used for production of other terpenoids in E. coli.

Background: Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory., Results: Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype., Conclusions: The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.

Background: Plasmid expression is a popular method in studies of MVA pathway for isoprenoid production in Escherichia coli. However, heterologous gene expression with plasmid is often not stable and might burden growth of host cells, decreases cell mass and product yield. In this study, MVA pathway was divided into three modules, and two heterologous modules were integrated into the E. coli chromosome. These modules were individually modulated with regulatory parts to optimize efficiency of the pathway in terms of downstream isoprenoid production., Results: MVA pathway modules Hmg1-erg12 operon and mvaS-mvaA-mavD1 operon were integrated into E. coli chromosome followed by modulation with promoters with varied strength. Along with activation of atoB, a 26% increase of β-carotene production with no effect on cell growth was obtained. With a combinatory modulation of two key enzymes mvas and Hmg1 with degenerate RBS library, β-carotene showed a further increase of 51%., Conclusions: Our study provides a novel strategy for improving production of a target compound through integration and modulation of heterologous pathways in both transcription and translation level. In addition, a genetically hard-coded chassis with both efficient MEP and MVA pathways for isoprenoid precursor supply was constructed in this work.

Background: Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods., Results: In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h., Conclusion: In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci.

Redox imbalance has been regarded as the key limitation for anaerobic isobutanol production in metabolically engineered Escherichia coli strains. In this work, the ethanol synthetic pathway was recruited to solve the NADH redundant problem while the pentose phosphate pathway was modulated to solve the NADPH deficient problem for anaerobic isobutanol production. Recruiting the ethanol synthetic pathway in strain AS108 decreased isobutanol yield from 0.66 to 0.29 mol/mol glucose. It was found that there was a negative correlation between aldehyde/alcohol dehydrogenase (AdhE) activity and isobutanol production. Decreasing AdhE activity increased isobutanol yield from 0.29 to 0.6 mol/mol. On the other hand, modulation of the glucose 6-phosphate dehydrogenase gene of the pentose phosphate pathway increased isobutanol yield from 0.29 to 0.41 mol/mol. Combination of these two strategies had a synergistic effect on improving isobutanol production. Isobutanol titer and yield of the best strain ZL021 were 53 mM and 0.74 mol/mol, which were 51 % and 12 % higher than the starting strain AS108, respectively. The total alcohol yield of strain ZL021 was 0.81 mol/mol, which was 23 % higher than strain AS108.

Quantification of energy and redox cofactors is of great value to synthetic biologists to infer the balance of energy metabolism in engineered microbial strains and assess each strain's potential for further improvement. Most currently used methods for intracellular cofactor measurement suffer from incomplete coverage, low reproducibility, suboptimal sensitivity or specificity. In this study, we described an SPE-HILIC/MS approach for simultaneous determination of six cofactor targets (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli cells. Sufficient linearity, precision and metabolite recoveries of this new approach justified its reliability in targeted cofactor quantification. Our approach was then compared with conventional enzymatic assays to demonstrate its superior performance. We applied the SPE-HILIC/MS approach to profile shift of cofactor balances in several engineered E. coli strains with varying isobutanol production. Our cofactor analysis clearly revealed that optimal energy fitness was achieved in the highest-yield strain through combined modulation of a transhydrogenase and a NAD(+) kinase. Apart from the targeted cofactors, the SPE enrichment procedure also allowed for confident identification of 39 groups of polar metabolites mainly involved in central carbon metabolism in E. coli cells., (Copyright © 2016. Published by Elsevier B.V.)

Production of bulk chemicals from renewable biomass has been proved to be sustainable and environmentally friendly. Escherichia coli is the most commonly used host strain for constructing cell factories for production of bulk chemicals since it has clear physiological and genetic characteristics, grows fast in minimal salts medium, uses a wide range of substrates, and can be genetically modified easily. With the development of metabolic engineering, systems biology, and synthetic biology, a technology platform has been established to construct E. coli cell factories for bulk chemicals production. In this chapter, we will introduce this technology platform, as well as E. coli cell factories successfully constructed for production of organic acids and alcohols.

Phenol is a bulk chemical with lots of applications in the chemical industry. Fermentative production of phenol had been realized in both Pseudomonas putida and Escherichia coli by recruiting tyrosine phenol-lyase (TPL). The TPL pathway needs tyrosine as the direct precursor for phenol production. In this work, a novel phenol synthetic pathway was created in E. coli by recruiting 4-hydroxybenzoate decarboxylase, which can convert 4-hydroxybenzoate to phenol and carbon dioxide. Activating 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and chorismate pyruvate lyase (UbiC) through plasmid overexpression led to 7- and 69-fold increase of phenol production, respectively, demonstrating that these two enzymes were the rate-limiting steps for phenol production. Genetically stable strains were then obtained by gene integration and gene modulation directly in chromosome. Phenol titer increased 147-fold (from 1.7 to 250 mg/L) after modulating the DAHP synthase, UbiC, and 4-hydroxybenzoate decarboxylase genes in chromosome. Five solvents were tested for two-phase extractive fermentation to eliminate phenol toxicity to E. coli cells. Tributyrin and dibutyl phthalate were the best two solvents for improving phenol production, leading to 23 and 30 % increase of total phenol production, respectively. Two-phase fed-batch fermentation of the best strain Phe009 was performed in a 7 L fermentor, which produced 9.51 g/L phenol with a yield of 0.061 g/g glucose.

An engineered strain for the conversion of D-fructose to allitol was developed by constructing a multi-enzyme coupling pathway and cofactor recycling system in Escherichia coli. D-Psicose-3-epimerase from Ruminococcus sp. and ribitol dehydrogenase from Klebsiella oxytoca were coexpressed to form the multi-enzyme coupling pathway for allitol production. The cofactor recycling system was constructed using the formate dehydrogenase gene from Candida methylica for continuous NADH supply. The recombinant strain produced 10.62 g/l allitol from 100 mM D-fructose. To increase the intracellular concentration of the substrate, the glucose/fructose facilitator gene from Zymomonas mobilis was incorporated into the engineered strain. The results showed that the allitol yield was enhanced significantly to 16.53 g/l with a conversion rate of 92 %. Through optimizing conversion conditions, allitol was produced effectively on a large scale by the whole-cell biotransformation system; the yield reached 48.62 g/l when 500 mM D-fructose was used as the substrate.

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.

Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.

β-carotene belongs to carotenoids family, widely applied in pharmaceuticals, neutraceuticals, cosmetics and food industries. In this study, three key genes (dxs, idi, and crt operon) within β-carotene synthetic pathway in recombinant Escherichia coli strain CAR005 were modulated with RBS Library to improve β-carotene production. There were 7%, 11% and 17% increase of β-carotene yield respectively after modulating dxs, idi and crt operon genes with RBS Library, demonstrating that modulating gene expression with regulatory parts libraries would have more opportunities to obtain optimal production of target compound. Combined modulation of crt operon, dxs and idi genes led to 35% increase of β-carotene yield compared to parent strain CAR005. The optimal gene expression strength identified in single gene modulation would not be the optimal strength when used in combined modulation. Our study provides a new strategy for improving production of target compound through modulation of gene expression.

Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5 mg lycopene/l (6.5 mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76 % increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32 %. The optimal strain LYC010 produced 3.52 g lycopene/l (50.6 mg/g DCW) in fed-batch fermentation.

Although many efforts had been performed to engineer Escherichia coli for succinate production, succinate efflux system had not been investigated as an engineering target for improving succinate production. In this work, four Dcu transporters, which had been reported to be responsible for C4-dicarboxylates transportation of E. coli, were investigated for their succinate efflux capabilities. These four dcu genes were deleted individually in a previously constructed succinate-producing strain to study their effects on succinate production. Deleting dcuA and dcuD genes had nearly no influence, while deleting dcuB and dcuC genes led to 15 and 11% decrease of succinate titer, respectively. Deleting both dcuB and dcuC genes resulted in 90% decrease of succinate titer, suggesting that DcuB and DcuC were the main transporters for succinate efflux and they functioned as independent and mutually redundant succinate efflux transporters. Furthermore, RBS library having strengths varied from 0.17 to 8.6 times of induced E. coli lacZ promoter was used to modulate dcuB and dcuC genes for improving succinate production. Modulating these two genes in combination led to 34% increase of succinate titer. To the best of knowledge, this was the first report about improving succinate production through engineering succinate efflux system.

Short branched-chain acyl-CoAs are important building blocks for a wide variety of pharmaceutically valuable natural products. Escherichia coli has been used as a heterologous host for the production of a variety of natural compounds for many years. In the current study, we engineered synthesis of isobutyryl-CoA and isovaleryl-CoA from glucose in E. coli by integration of the branched-chain α-keto acid dehydrogenase complex from Streptomyces avermitilis. In the presence of the chloramphenicol acetyltransferase (cat) gene, chloramphenicol was converted to both chloramphenicol-3-isobutyrate and chloramphenicol-3-isovalerate by the recombinant E. coli strains, which suggested successful synthesis of isobutyryl-CoA and isovaleryl-CoA. Furthermore, we improved the α-keto acid precursor supply by overexpressing the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E. coli and thus enhanced the synthesis of short branched-chain acyl-CoAs. By feeding 25 mg/L chloramphenicol, 2.96 ± 0.06 mg/L chloramphenicol-3-isobutyrate and 3.94 ± 0.06 mg/L chloramphenicol-3-isovalerate were generated by the engineered E. coli strain, which indicated efficient biosynthesis of short branched-chain acyl-CoAs. HPLC analysis showed that the most efficient E. coli strain produced 80.77 ± 3.83 nmol/g wet weight isovaleryl-CoA. To our knowledge, this is the first report of production of short branched-chain acyl-CoAs in E. coli and opens a way to biosynthesize various valuable natural compounds based on these special building blocks from renewable carbon sources.

D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.

Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1g/L β-carotene with a yield of 60mg/g DCW., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic isobutanol production. This strategy will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways. In addition, combined activation of PntAB and YfjB led to 28% and 22% increase of aerobic isobutanol titer and yield, resulting in production of 10.8g/L isobutanol in 24h with a yield of 0.62mol/mol., (Copyright © 2012 Elsevier Inc. All rights reserved.)