Efficient biosynthesis of 3-hydroxypropionic acid from ethanol in metabolically engineered Escherichia coli.
Bioresource technology
Engineering microbial cell factories to convert CO-based feedstock into chemicals and fuels provide a feasible carbon-neutral route for the third-generation biorefineries. Ethanol became one of the major products of syngas fermentation by engineered acetogens. The key building block chemical 3-hydroxypropionic acid (3-HP) can be synthesized from ethanol by the malonyl-CoA pathway with CO fixation. In this study, the effect of two ethanol consumption pathways on 3-HP synthesis were studied as well as the effect of TCA cycle, gluconeogenesis pathway, and transhydrogenase. And the 3-HP synthesis pathway was also optimized. The engineered strain synthesized 1.66 g/L of 3-HP with a yield of 0.24 g/g. Furthermore, the titer and the yield of 3-HP increased to 13.17 g/L and 0.57 g/g in the whole-cell biocatalysis system. This study indicated that ethanol as feedstock had the potential to synthesize 3-HP, which provided an alternative route for future biorefinery.
10.1016/j.biortech.2022.127907
Production of 3-hydroxypropionic acid from acetate using metabolically-engineered and glucose-grown Escherichia coli.
Lama Suman,Kim Yeonhee,Nguyen Dat Tuan,Im Chae Ho,Sankaranarayanan Mugesh,Park Sunghoon
Bioresource technology
Acetate can be used as carbon feedstock for the production of 3-hydroxypropionic acid (3-HP), but the production level was low due to inefficient cell growth on acetate. To better utilize acetate, a two-stage strategy, whereby glucose is used for cell growth and acetate for 3-HP formation, was attempted. Dissected malonyl-CoA reductase of Chloroflexus aurantiacus, alone or along with acetyl-CoA carboxylase and/or transhydrogenase, was overexpressed, and by-products formation pathway, glyoxylate shunt (GS) and gluconeogenesis were modified. When GS or gluconeogenesis was disrupted, cell growth on glucose was not hampered, while on acetate it was completely abolished. Consequently, 3-HP production, at production stage, were low, though 3-HP yield on acetate was increased, especially in the case of aceA deletion. In two-stage bioreactor, strain with upregulated GS produced 7.3 g/L 3-HP with yield of 0.26 mol/mol acetate. This study suggests that two-stage cultivation is a good strategy for 3-HP production from acetate.
10.1016/j.biortech.2020.124362
Engineering E. coli for the biosynthesis of 3-hydroxy-γ-butyrolactone (3HBL) and 3,4-dihydroxybutyric acid (3,4-DHBA) as value-added chemicals from glucose as a sole carbon source.
Dhamankar Himanshu,Tarasova Yekaterina,Martin Collin H,Prather Kristala L J
Metabolic engineering
3-hydroxy-γ-butyrolactone (3HBL) is a versatile chiral synthon, deemed a top value-added chemical from biomass by the DOE. We recently reported the first biosynthetic pathway towards 3HBL and its hydrolyzed form, 3,4-dihydroxybutyric acid (3,4-DHBA) in recombinant Escherichia coli using glucose and glycolic acid as feedstocks and briefly described their synthesis solely from glucose. Synthesis from glucose requires integration of the endogenous glyoxylate shunt with the 3,4-DHBA/3HBL pathway and co-overexpression of seven genes, posing challenges with respect to expression, repression of the glyoxylate shunt and optimal carbon distribution between the two pathways. Here we discuss engineering this integration. While appropriate media and over-expression of glyoxylate shunt enzymes helped overcome repression, two orthogonal expression systems were employed to address the expression and carbon distribution challenge. Synthesis of up to 0.3g/L of 3HBL and 0.7g/L of 3,4-DHBA solely from glucose was demonstrated, amounting to 24% of the theoretical maximum.
10.1016/j.ymben.2014.06.004
[Improving glycolic acid yield by metabolic engineering in Escherichia coli].
Ma Ning,Zhu Kangjia,Mao Yin,Deng Yu
Sheng wu gong cheng xue bao = Chinese journal of biotechnology
Glycolic acid is an important industrial compound. To improve glycolic acid yield, we knocked out ldhA (lactate dehydrogenase) in Escherichia coli MG1655 (DE3) to get the strain Mgly1. Then, we regulated expression levels of isocitrate lyase (aceA), glyoxylic acid reductase (ycdW) and isocitrate dehydrogenase kinase/phosphorylase (aceK) that are key enzymes of glycolate synthesis pathway. The yield of glycolic acid increased to 0.326 g/g glucose (38.3% of the theoretical yield) by overexpressing citrate synthase (gltA). Then we knocked out glcB and aceB (malate synthase) in Mgly1. The engineering strain Mgly335 was obtained and the yield of glycolic acid reached 0.522 g/g glucose (61.4% of the theoretical yield).
10.13345/j.cjb.170209
Engineering Escherichia coli for glycolic acid production from D-xylose through the Dahms pathway and glyoxylate bypass.
Cabulong Rhudith B,Lee Won-Keun,Bañares Angelo B,Ramos Kristine Rose M,Nisola Grace M,Valdehuesa Kris Niño G,Chung Wook-Jin
Applied microbiology and biotechnology
Glycolic acid (GA) is an ⍺-hydroxy acid used in cosmetics, packaging, and medical industries due to its excellent properties, especially in its polymeric form. In this study, Escherichia coli was engineered to produce GA from D-xylose by linking the Dahms pathway, the glyoxylate bypass, and the partial reverse glyoxylate pathway (RGP). Initially, a GA-producing strain was constructed by disrupting the xylAB and glcD genes in the E. coli genome and overexpressing the xdh(Cc) from Caulobacter crescentus. This strain was further improved through modular optimization of the Dahms pathway and the glyoxylate bypass. Results for module 1 showed that the rate-limiting step of the Dahms pathway was the xylonate dehydratase reaction, and the overexpression of yagF was sufficient to overcome this bottleneck. Furthermore, the appropriate aldolase gene for module 1 was proven to be yagE. The results also show that overexpression of the lactaldehyde dehydrogenase gene, aldA, is needed to increase the GA production while the overexpression of glyoxylate reductase gene, ycdW, was only essential when the glyoxylate bypass was active. On the other hand, the module 2 enzymes AceA and AceK were vital in activating the glyoxylate bypass, while the RGP enzymes were dispensable. The final strain (GA19) produced 4.57 g/L GA with a yield of 0.46 g/g from D-xylose. So far, this is the highest value achieved for GA production in engineered E. coli through the Dahms pathway.
10.1007/s00253-018-8744-8
Autonomous production of 1,4-butanediol via a de novo biosynthesis pathway in engineered Escherichia coli.
Liu Huaiwei,Lu Ting
Metabolic engineering
1,4-Butanediol (BD) is an important chemical that is widely used in industry with an annual demand of one million metric tons. Here we report a modular development of engineered bacteria for successful BD bio-production. Using a systems engineering concept, we partitioned our development into two parts: namely BD biosynthesis and production control. The former was implemented through a de novo pathway that functions as an enzymatic reactor, while the latter was accomplished via synthetic circuits serving as genetic controllers. To facilitate development, the carbon utilizations were also partitioned into two routes. d-xylose was exclusively designated for BD production with other carbon sources utilized for cellular growth. Additionally, a quorum-sensing mechanism was exploited for production control, and the resulting strain was capable of autonomous production of BD. This study represents an example of the synergy between synthetic biology and metabolic engineering, affirming the need for deeper integration of the two fields.
10.1016/j.ymben.2015.03.009
Combining CRISPR and CRISPRi Systems for Metabolic Engineering of E. coli and 1,4-BDO Biosynthesis.
Wu Meng-Ying,Sung Li-Yu,Li Hung,Huang Chun-Hung,Hu Yu-Chen
ACS synthetic biology
Biosynthesis of 1,4-butanediol (1,4-BDO) in E. coli requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of E. coli and 1,4-BDO production. We first exploited CRISPR to perform point mutation of gltA, replacement of native lpdA with heterologous lpdA, knockout of sad and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (cat1, sucD, 4hbd, cat2, bld, bdh) in the 1,4-BDO biosynthesis pathway. The successive E. coli engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (gabD, ybgC and tesB) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as E. coli and production of chemicals (e.g., 1,4-BDO).
10.1021/acssynbio.7b00251
Achievements and Perspectives in 1,4-Butanediol Production from Engineered Microorganisms.
Cheng Jie,Li Juan,Zheng Linggang
Journal of agricultural and food chemistry
1,4-Butanediol (1,4-BDO), a significant commodity chemical, is currently manufactured exclusively from a host of energy-intensive processes, accompanied by severe environmental issues, such as the greenhouse effect and air pollution. As a result of the ever-increasing global market demands and increasing applications of 1,4-BDO, attention has turned to the sustainable bioproduction of 1,4-BDO, and several bio-based approaches for 1,4-BDO production have been successfully established in engineered , including biosynthesis and biocatalysis. Recent achievements in enhancing the accumulation of 1,4-BDO have been achieved by metabolic engineering strategies, such as improving precursor supply, enhancing activities of critical enzymes, and fewer byproduct synthesis. Here, we summarize the primary advances of the biological pathway for 1,4-BDO synthesis and put forward the future development prospect of bio-based 1,4-BDO production.
10.1021/acs.jafc.1c03769
Functional genomic assessment of 2, 2-bis (bromomethyl)-1, 3-propanediol induced cytotoxicity in a single-gene knockout library of E. coli.
Guan Miao,Zhang Xiaowei
Chemosphere
Functional gene fingerprinting of chemicals could be used to understand the direct gene-chemical interaction in the process of toxification from a genome-wide scale. 2, 2-bis (bromomethyl)-1, 3-propanediol (BMP) is a brominated flame retardant with widespread production but with very limited toxicological data. Here the cytotoxicity of BMP was assessed by Escherichia coli (E. coli) functional genome-wide knockout mutants screening and the underlying molecular mechanism was investigated. The median inhibition concentration (IC50) of BMP was 1.608 ± 0.078 mg/ml after 24 h exposure. 119 initial, including 66 sensitive and 53 resistant single gene mutants, were identified by a full library screening of BMP at the concentration of IC50. The resistant genes were significantly enriched in nucleobase-containing compound biosynthetic process (GO: 0034654) by gene ontology (GO) biological process analyses, which suggested that the pathway of DNA repair is a critical cellular process in the survival of cells exposed to BMP. Meanwhile, function annotation of all BMP responsive genes suggested the mechanism of BMP was associated with DNA damage, oxidative stress and cellular transmembrane transport process. Many genes were exclusively responsive to BMP comparing with other chemicals that has been assessed by E. coli mutant screening approach, which indicated that BMP has a distinct mode of toxic action. Overall, the functional genomic screening approach presented here provides a great tool to assess the cellular toxicological mechanism of environmental chemicals.
10.1016/j.chemosphere.2017.07.031
Glycerol Dehydratases: Biochemical Structures, Catalytic Mechanisms, and Industrial Applications in 1,3-Propanediol Production by Naturally Occurring and Genetically Engineered Bacterial Strains.
Liu Jian-Zhong,Xu Wu,Chistoserdov Andrei,Bajpai Rakesh K
Applied biochemistry and biotechnology
To date, two types of glycerol dehydratases have been reported: coenzyme B12-dependent and coenzyme B12-independent glycerol dehydratases. The three-dimensional structure of the former is a dimer of αβγ heterotrimer, while that of the latter is a homodimer. Their mechanisms of reaction are typically enzymatic radical catalysis. Functional radical in both the glycerol dehydratases is the adenosyl radical. However, the adenosyl radical in the former originates from coenzyme B12 by homolytic cleavage, and that in the latter from S-adenosyl-methionine. Until some years ago, Clostridium butyricum VPI 1718 was the only microorganism known to possess B12-independent glycerol dehydratase, but since then, several other bacteria with this unique capability have been identified. This article focuses on the glycerol dehydratases and on 1,3-propanediol production from glycerol by naturally occurring and genetically engineered bacterial strains containing glycerol dehydratase.
10.1007/s12010-016-2051-6
Engineering Escherichia coli to assimilate β-alanine as a major carbon source.
Applied microbiology and biotechnology
The threat of global plastic waste accumulation has spurred the exploration of plastics derived from biological sources. A well-known example is polyester made of 1,3-propanediol (1,3-PDO). However, there is no known pathway to assimilate 1,3-PDO into the central carbon metabolism, posing a potential challenge to upcycling such plastic wastes. Here, we proposed that the 1,3-PDO assimilation pathway could pass through malonate semialdehyde (MSA) as an intermediate. Since MSA is a toxic aldehyde, β-alanine was chosen as a surrogate substrate in this study to construct the lower part of the proposed pathway. To this end, we successfully engineered E. coli MG1655 to assimilate β-alanine as the major carbon source. β-alanine could be easily converted into MSA using a β-alanine/pyruvate transaminase from Pseudomonas aeruginosa (PaBapt). However, the subsequent step to generate acetyl-CoA from MSA was unknown. After a series of phenotype screenings, adaptive laboratory evolution and transcriptomic analysis, two CoA-acylating MSA dehydrogenases from Vibrio natriegens (VnMmsD), were found to be able to complete the metabolic pathway. Optical density at 600 nm (OD) of the resulting strain E. coli BA02 could reach 4.5 after 96 h. Two approaches were subsequently used to improve its performance. First, PaBapt and both VnMmsDs were expressed from a single plasmid to mitigate antibiotic stress. Second, a native 3-hydroxy acid dehydrogenase (EcYdfG) was disrupted to address the carbon loss to 3-hydroxypropionate (3-HP) production from MSA. OD of the best-performing strain E. coli BA07∆ could reach 6 within 24 h using 5 g/L β-alanine. The construction of E. coli BA07∆ lays a solid foundation to establishing a 1,3-PDO assimilation pathway. KEYPOINTS: • This study demonstrates the implementation of a metabolic pathway to assimilate β-alanine as the major carbon source in E. coli MG1655. • Two V. natriegens CoA-acylating methyl malonate semialdehyde dehydrogenases were used to complete the pathway in E. coli BA02. • The construction of E. coli BA02 also revealed the plasmid fusion event between two plasmids with the same replication origin.
10.1007/s00253-023-12574-5
High-level co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Metabolic engineering and process optimization.
Bioresource technology
3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) are value-added chemicals with versatile applications in the chemical, pharmaceutical, and food industries. Nevertheless, sustainable production of 3-HP and 1,3-PDO is often limited by the lack of efficient strains and suitable fermentation configurations. Herein, attempts have been made to improve the co-production of both metabolites through metabolic engineering of Escherichia coli and process optimization. First, the 3-HP and 1,3-PDO co-biosynthetic pathways were recruited and optimized in E. coli, followed by coupling the pathways to the transhydrogenase-mediated cofactor regeneration systems that increased cofactor availability and product synthesis. Next, pathway rebalancing and block of by-product formation significantly improved 3-HP and 1,3-PDO net titer. Subsequently, glycerol flux toward 3-HP and 1,3-PDO synthesis was maximized by removing metabolic repression and fine-tuning the glycerol oxidation pathway. Lastly, the combined fermentation process optimization and two-stage pH-controlled fed-batch fermentation co-produced 140.50 g/L 3-HP and 1,3-PDO, with 0.85 mol/mol net yield.
10.1016/j.biortech.2022.128438
Engineering Escherichia coli for Direct Production of 1,2-Propanediol and 1,3-Propanediol from Starch.
Sato Rintaro,Tanaka Tomonari,Ohara Hitomi,Aso Yuji
Current microbiology
Diols are versatile chemicals used for multiple manufacturing products. In some previous studies, Escherichia coli has been engineered to produce 1,2-propanediol (1,2-PDO) and 1,3-propanediol (1,3-PDO) from glucose. However, there are no reports on the direct production of these diols from starch instead of glucose as a substrate. In this study, we directly produced 1,2-PDO and 1,3-PDO from starch using E. coli engineered for expressing a heterologous α-amylase, along with the expression of 1,2-PDO and 1,3-PDO synthetic genes. For this, the recombinant plasmids, pVUB3-SBA harboring amyA gene for α-amylase production, pSR5 harboring pct, pduP, and yahK genes for 1,2-PDO production, and pSR8 harboring gpd1-gpp2, dhaB123, gdrAB, and dhaT genes for 1,3-PDO production, were constructed. Subsequently, E. coli BW25113 (ΔpflA) and BW25113 strains were transformed with pVUB3-SBA, pSR5, and/or pSR8. Using these transformants, direct production of 1,2-PDO and 1,3-PDO from starch was demonstrated under microaerobic condition. As a result, the maximum production titers of 1,2-PDO and 1,3-PDO from 1% glucose as a sole carbon source were 13 mg/L and 150 mg/L, respectively. The maximum production titers from 1% starch were similar levels (30 mg/L 1,2-PDO and 120 mg/L 1,3-PDO). These data indicate that starch can be an alternative carbon source for the production of 1,2-PDO and 1,3-PDO in engineered E. coli. This technology could simplify the upstream process of diol bioproduction.
10.1007/s00284-020-02189-8