A citric acid cycle-deficient Escherichia coli as an efficient chassis for aerobic fermentations.

Tricarboxylic acid cycle (TCA cycle) plays an important role for aerobic growth of heterotrophic bacteria. Theoretically, eliminating TCA cycle would decrease carbon dissipation and facilitate chemicals biosynthesis. Here, we construct an E. coli strain without a functional TCA cycle that can serve as a versatile chassis for chemicals biosynthesis. We first use adaptive laboratory evolution to recover aerobic growth in minimal medium of TCA cycle-deficient E. coli. Inactivation of succinate dehydrogenase is a key event in the evolutionary trajectory. Supply of succinyl-CoA is identified as the growth limiting factor. By replacing endogenous succinyl-CoA dependent enzymes, we obtain an optimized TCA cycle-deficient E. coli strain. As a proof of concept, the strain is engineered for high-yield production of four separate products. This work enhances our understanding of the role of the TCA cycle in E. coli metabolism and demonstrates the advantages of using TCA cycle-deficient E. coli strain for biotechnological applications.

Physical Activity among Rural Residents in Eastern, Central, and Western Provinces of China: A Cross-Sectional Survey.

Physical activity (PA) in which physical exercise (PE) is an important component is probably the most important intervention for preventing noncommunicable diseases (NCDs). However, few studies on PA and PE of rural residents in China were reported. This study conducted the first population-based cross-sectional survey in three provinces of China in 2021 that examined both PA and PE as well as the associated factors of rural residents. The International Physical Activity Questionnaire Short Form (IPAQ-S) was used, and a total of 3780 rural residents were surveyed. The result showed that 22.2% of the rural residents were physical inactivity and rural residents reporting practice of PE was 54.4%. Binary logistic regression analyses showed that being female, people aged between 15 to 34 years or 60 years old and above, employees of governmental departments/retirees, school students, the unemployed, and people with NCDs were risk factors of PA while ethnic minority groups, smoking, and alcohol consumption were risk factors of PE. Health promotion programme aiming at increasing people's PA in rural China is urgently needed, and it should focus on the population groups of the female, people aged 60 years and above, school students, the unemployed, and people with NCDs.

Improved methylation in E. coli via an efficient methyl supply system driven by betaine.

Methylation reactions are involved in the biosynthesis of various natural molecules, in which S-adenosyl-L-methionine (SAM) acts as the principal biological methyl donor. The limited availability of SAM often affects the biosynthesis of methylated metabolites in cells, especially when heterologous SAM-mediated methyltransferases are employed. To solve this problem, a methyl supply system driven by betaine was developed in this study to enhance SAM availability in cells. A reconstructed methionine cycle was designed in E. coli using betaine as the methyl source by introducing betaine-homocysteine methyltransferase. Ferulic acid served as a model product was used to test the efficiency of methyl supply system. ATP is a co-factor for SAM biosynthesis and a pathway for ATP regeneration from adenosine was introduced to maintain the stability of the adenylate pool. After testing two different S-adenosyl-L-homocysteine (SAH) hydrolysis pathways, the optimized SAHase pathway was adopted for converting SAH back to homocysteine (Hcy). Thus, a methyl supply system was developed which increased SAM availability and therefore improved the titer and productivity of ferulic acid by 12.6-fold and 15.9-fold, respectively. The system was also applied successfully for other methyltransferase-catalyzed reactions. This work provides an efficient methyl supply system for enhanced production of methylated chemicals using betaine as the methyl source.

Mask-Wearing and Handwashing Behaviors of Chinese Rural Residents during the Pandemic of COVID-19: A Cross-Sectional Survey.

OBJECTIVE: To understand mask-wearing and handwashing behaviors of Chinese rural residents during the COVID-19 pandemic and to analyze the associated factors. METHODS: This study used a multi-stage random sampling method to conduct a cross-sectional questionnaire survey during the period of July to December of 2021, in six counties located in Shandong, Shanxi, and Yunnan provinces representing the eastern, central, and western regions of China, respectively. A total of 3864 villagers were surveyed with a questionnaire, and 3832 valid questionnaires were finally analyzed. Descriptive statistics and logistic regression analysis were used for statistical analysis. RESULTS: Around ninety-four percent (93.6%) of rural residents reported mask-wearing during the COVID-19 pandemic, but only 44.5% of them could replace masks in time. Multivariate logistic regression analysis showed that those who were female, aged 15-59, had an education level of high school and above, were divorced/widowed, worked as farmers (workers), or were rural residents in Shandong Province were more likely to wear masks. Furthermore, those who were female, aged 15-59, had an education level of high school and above, were unmarried and married, were business and service workers, or were rural residents in Shandong and Shanxi Province replaced masks more timely. Around seventy percent (69.7%) of rural residents reported using soap when washing their hands, but only 38.0% of rural residents could wash their hands properly. Multivariate logistic regression analysis showed that rural residents who were aged 35-59, had an education of high school and above, or lived in Shandong Province and Shanxi Province were more likely to wash their hands with soap. Those who were aged 15-59, had an education of high school and above, worked as farmers (workers), were employees of governmental departments and retirees, were business and service workers, or were students had higher proper handwashing rates. CONCLUSION: During the COVID-19 pandemic, the proportion of Chinese rural residents wearing masks reached 93.6%, but only 44.5% were able to replace masks in time, gender, age, education level, marital status, occupation, and living place had an impact on mask-wearing. The proportion of Chinese rural residents who could wash hands with soap reached 69.7%, but only 38.0% could wash their hands properly. Age and education level were influencing factors for both washing-hand with soap and proper handwashing.

Highly Efficient Production of Menaquinone-7 from Glucose by Metabolically Engineered Escherichia coli.

Menaquinone-7 (MK-7) possesses wide health and medical value, and the market demand for MK-7 has increased. Metabolic engineering for MK-7 production in Escherichia coli still remains challenging due to the characteristics of the competing quinone synthesis, and cells mainly synthesized menaquinones under anaerobic conditions. To increase the production of MK-7 in engineered E. coli strains under aerobic conditions, we divided the whole MK-7 biosynthetic pathway into three modules (MVA pathway, DHNA pathway, and MK-7 pathway) and systematically optimized each of them. First, by screening and enhancing Idi expression, the amounts of MK-7/DMK-7 increased significantly. Then, in the MK-7 pathway, by combinatorial overexpression of endogenous MenA and exogenous UbiE, and fine-tuning the expression of HepPPS, MenA, and UbiE, 70 µM MK-7 was achieved. Third, the DHNA synthetic pathway was enhanced, and 157 µM MK-7 was achieved. By the combinational metabolic engineering strategies and membrane engineering, an efficient metabolic engineered E. coli strain for MK-7 synthesis was developed, and 200 µM (129 mg/L) MK-7 was obtained in shake flask experiment, representing a 306-fold increase compared to the starting strain. In the scale-up fermentation, 2074 µM (1350 mg/L) MK-7 was achieved after 52 h fermentation with a productivity of 26 mg/L/h. This is the highest titer of MK-7 ever reported. This study offers an alternative method for MK-7 production from biorenewable feedstock (glucose) by engineered E. coli. The high titer of our process should make it a promising cost-effective resource for MK-7.

Bioretrosynthesis of Functionalized N-Heterocycles from Glucose via One-Pot Tandem Collaborations of Designed Microbes.

The design of multistrain systems has markedly expanded the prospects of using long biosynthetic pathways to produce natural compounds. However, the cooperative use of artificially engineered microbes to synthesize xenobiotic chemicals from renewable carbohydrates is still in its infancy. Here, a microbial system is developed for the production of high-added-value N-heterocycles directly from glucose. Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5-methyl-2-pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. Via one-pot tandem collaborations, the designed microbes remarkably realize high-level production of 5-methyl-2-pyrazinecarboxylic acid (6.2 ± 0.1 g L-1) and its precursor 2,5-dimethylpyrazine (7.9 ± 0.7 g L-1). This study is the first application of cooperative microbes for the total biosynthesis of functionalized N-heterocycles and provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses.

Menaquinone-7 production in engineered Escherichia coli.

Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. In this study, to develop engineered Escherichia coli strains for MK-7 production, heterogeneous heptaprenyl pyrophosphate synthetase (HepPPS) was introduced, and MK-7 production was first achieved in engineered E. coli by overexpression of Bacillus subtilis-derived HepPPS (BsHepPPS). Then, by optimizing the enzyme expression of the heterogenous mevalonic acid (MVA) pathway and the BsHepPPS, the titre of MK-7 increased to 2.3 µM, which was 22-fold higher than that of the original strain. The competitive pathways of MK-7 were further investigated by deletion of ubiCA or ispB. Finally, the scale-up fermentation of the engineered E. coli in a 5-L fermenter was studied under aerobic conditions using glucose, and 13.6 µM (8.8 mg/L) MK-7 was achieved. Additionally, metabolite analysis revealed a new bottleneck in the MK-7 pathway at ubiE, suggesting an avenue for further optimization. This report is the first to describe the metabolic engineering of MK-7 in E. coli, which provides a new perspective for MK-7 production.

Reconstitution of the Ornithine Cycle with Arginine:Glycine Amidinotransferase to Engineer Escherichia coli into an Efficient Whole-Cell Catalyst of Guanidinoacetate.

Guanidino compounds can be synthesized by transamidination reactions using arginine as a guanidine group donor. The efficiency of guanidino biosynthesis is often affected by the supply of arginine and the inhibition of the coproduct ornithine. To alleviate this shortcoming, we designed a reconstituted ornithine cycle in Escherichia coli to engineer an efficient whole-cell catalyst for guanidinoacetate (GAA) production by introducing a heterogeneous arginine:glycine amidinotransferase (AGAT). To alleviate the inhibition of ornithine, a citrulline synthetic module was constructed and optimized by introducing a glutamine self-sufficient system. Then, to improve the pathway from citrulline to arginine, an aspartate self-sufficient system was introduced into the arginine synthetic module. By combining these modules (GAA, citrulline, and arginine synthetic modules), a reconstituted ornithine cycle was developed, which significantly improved the biocatalyst efficiency (3.9-fold increase). In the system, arginine was regenerated efficiently through the reconstituted ornithine cycle, which converted arginine from a substrate to a cofactor for the transamidination reaction, thereby relieving the ornithine inhibition. Moreover, the amidino group of GAA in this system was mainly supplied by carbon and nitrogen assimilation. After the engineering process, 8.61 g/L GAA (73.56 mM) with a productivity of 0.39 g/L/h was achieved in a 22 h bioconversion. To the best of our knowledge, this is the first time that GAA has been produced in E. coli. This reconstructed ornithine cycle could be used as a transamidination platform for amidino group supply and has potential applications in the biosynthesis of other guanidino compounds.

Metabolic engineering of Escherichia coli for efficient production of L-alanyl-L-glutamine.

BACKGROUND: L-Alanyl-L-glutamine (AQ) is a functional dipeptide with high water solubility, good thermal stability and high bioavailability. It is widely used in clinical treatment, post-operative rehabilitation, sports health care and other fields. AQ is mainly produced via chemical synthesis which is complicated, time-consuming, labor-intensive, and have a low yield accompanied with the generation of by-products. It is therefore highly desirable to develop an efficient biotechnological process for the industrial production of AQ. RESULTS: A metabolically engineered E. coli strain for AQ production was developed by over-expressing L-amino acid α-ligase (BacD) from Bacillus subtilis, and inactivating the peptidases PepA, PepB, PepD, and PepN, as well as the dipeptide transport system Dpp. In order to use the more readily available substrate glutamic acid, a module for glutamine synthesis from glutamic acid was constructed by introducing glutamine synthetase (GlnA). Additionally, we knocked out glsA-glsB to block the first step in glutamine metabolism, and glnE-glnB involved in the ATP-dependent addition of AMP/UMP to a subunit of glutamine synthetase, which resulted in increased glutamine supply. Then the glutamine synthesis module was combined with the AQ synthesis module to develop the engineered strain that uses glutamic acid and alanine for AQ production. The expression of BacD and GlnA was further balanced to improve AQ production. Using the final engineered strain p15/AQ10 as a whole-cell biocatalyst, 71.7 mM AQ was produced with a productivity of 3.98 mM/h and conversion rate of 71.7%. CONCLUSION: A metabolically engineered strain for AQ production was successfully developed via inactivation of peptidases, screening of BacD, introduction of glutamine synthesis module, and balancing the glutamine and AQ synthesis modules to improve the yield of AQ. This work provides a microbial cell factory for efficient production of AQ with industrial potential.

Metabolic engineering for efficient supply of acetyl-CoA from different carbon sources in Escherichia coli.

BACKGROUND: Acetyl-CoA is an important metabolic intermediate and serves as an acetylation precursor for the biosynthesis of various value-added acetyl-chemicals. Acetyl-CoA can be produced from glucose, acetate, or fatty acids via metabolic pathways in Escherichia coli. Although glucose is an efficient carbon source for acetyl-CoA production, the pathway from acetate to acetyl-CoA is the shortest and fatty acids can produce acetyl-CoA through fatty acid oxidation along with abundant NADH and FADH2. In this study, metabolically engineered E. coli strains for efficiently supplying acetyl-CoA from glucose, acetate, and fatty acid were constructed and applied in one-step biosynthesis of N-acetylglutamate (NAG) from glutamate and acetyl-CoA. RESULTS: A metabolically engineered E. coli strain for NAG production was constructed by overexpressing N-acetylglutamate synthase from Kitasatospora setae in E. coli BW25113 with argB and argA knockout. The strain was further engineered to utilize glucose, acetate, and fatty acid to produce acetyl-CoA. When glucose was used as a carbon source, the combined mutants of ∆ptsG::glk, ∆galR::zglf, ∆poxB::acs, ∆ldhA, and ∆pta were more efficient for supplying acetyl-CoA. The acetyl-CoA synthetase (ACS) pathway and acetate kinase-phosphate acetyltransferase (ACK-PTA) pathway from acetate to acetyl-CoA were investigated, and the ACK-PTA pathway showed to be more efficient for supplying acetyl-CoA. When fatty acid was used as a carbon source, acetyl-CoA supply was improved by deletion of fadR and constitutive expression of fadD under the strong promoter CPA1. Comparison of acetyl-CoA supply from glucose, acetate and palmitic acid revealed that a higher conversion rate of glutamate (98.2%) and productivity (an average of 6.25 mmol/L/h) were obtained when using glucose as a carbon source. The results also demonstrated the great potential of acetate and fatty acid to supply acetyl-CoA, as the molar conversion rate of glutamate was more than 80%. CONCLUSIONS: Metabolically engineered E. coli strains were developed for NAG production. The metabolic pathways of acetyl-CoA from glucose, acetate, or fatty acid were optimized for efficient acetyl-CoA supply to enhance NAG production. The metabolic strategies for efficient acetyl-CoA supply used in this study can be exploited for other chemicals that use acetyl-CoA as a precursor or when acetylation is involved.

Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative ß-alanine with high stoichiometric yield.

L-aspartate is an important 4-carbon platform compound that can be used as the precursor of numerous chemical products. The bioproduction of L-aspartate directly from biomass resources is expected to provide a more cost-competitive technique route. Yet little metabolic engineering work on this matter has been carried out. In this study, we designed a shortcut pathway of L-aspartate biosynthesis in Escherichia coli, with a maximized stoichiometric yield of 2 mol/mol glucose. L-aspartate aminotransferase (AspC) was overexpressed for producing L-aspartate and coexpressed with L-aspartate-a-decarboxylase (PanD) for producing L-aspartate's derivative ß-alanine. L-aspartate could only be detected after directing carbon flux towards oxaloacetate and blocking the "futile cycle" with TCA cycle. A cofactor self-sufficient system successfully improved the efficiency of AspC-catalyzing L-aspartate biosynthesis reaction, and the glucose uptake remolding capably decreased byproducts from pyruvate. More targets were modified for relieving the bottleneck during fed-batch bioconversion. As a result, 1.01 mol L-aspartate/mol glucose and 1.52 mol ß-alanine/mol glucose were produced in corresponding strains respectively. Fed-batch bioconversion allowed 249 mM (33.1 g/L) L-aspartate or 424 mM (37.7 g/L) ß-alanine production, respectively. The study provides a novel promising metabolic engineering route for the production of L-aspartate and its derivate chemicals using biomass resources. These results also represent the first report of the efficient bioproduction of L-aspartate directly from glucose in E. coli and the highest yield of ß-alanine reported so far.

Cofactor self-sufficient whole-cell biocatalysts for the production of 2-phenylethanol.

The efficiency of biocatalysis is often affected by an insufficient supply and regeneration of cofactors and redox equivalents. To alleviate this shortcoming, a cofactor self-sufficient system was developed for enhanced production of 2-phenylethanol (2-PE) in E. coli. A "bridge" between the amino acid and its corresponding alcohol was designed in the system using glutamate dehydrogenase. By coupling glutamate dehydrogenase with transaminase and alcohol dehydrogenase, the cosubstrate (2-oxoglutarate) and redox equivalents (NAD(P)H) were regenerated simultaneously, so that no external cofactor or redox source was required. Thus, a cofactor self-sufficient system was developed, which improved the biocatalyst efficiency 3.8-fold. The ammonium generated in this process was removed using zeolite, which further improved the biosynthetic efficiency and resulted in a cleaner system. To the best of our knowledge, this system yielded the highest titer of 2-PE ever obtained in E. coli. Additionally, the wider applicability of this self-sufficient strategy was demonstrated in the production of D-phenyllactic acid. This study thus offers a new method to resolve the cofactor/redox imbalance problem and demonstrates the feasibility of the cofactor self-sufficient strategy for enhanced production of diverse chemicals.

Synthesis of Sialic Acids, Their Derivatives, and Analogs by Using a Whole-Cell Catalyst.

Sialic acids (Sias) are important constituents of cell surface glycans. Ready access to Sias in large quantities would facilitate the development of carbohydrate-based vaccines and small-molecule drugs. We now present a facile method for synthesizing various natural forms and non-natural derivatives or analogs of Sias by using a whole-cell catalyst, which is constructed by adding a plasmid containing necessary enzyme genes into a metabolically engineered strain of Escherichia coli. The flexible substrate tolerance of incorporated enzymes (N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid aldolase) allows the cellular catalyst to convert a wide range of simple and inexpensive sugars into various Sia-related compounds through an easily scalable fermentation process. Further, syntheses using this whole-cell biotransformation in combination with three conventional enzymatic reactions provide a series of complex Sia-containing glycans (sialyloligosaccharides) and their derivatives bearing different substituents. The processes described herein should permit the large-scale and economical production of both Sias and sialyloligosaccharides, and may complement existing chemical and enzymatic strategies.

Whole-cell biocatalysts by design.

Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.

Engineering deacetoxycephalosporin C synthase as a catalyst for the bioconversion of penicillins.

7-aminodeacetoxycephalosporanic acid (7-ADCA) is a key intermediate of many clinically useful semisynthetic cephalosporins that were traditionally prepared by processes involving chemical ring expansion of penicillin G. Bioconversion of penicillins to cephalosporins using deacetoxycephalosporin C synthase (DAOCS) is an alternative and environmentally friendly process for 7-ADCA production. Arnold Demain and co-workers pioneered such a process. Later, protein engineering efforts to improve the substrate specificity and catalytic efficiency of DAOCS for penicillins have been made by many groups, and a whole cell process using Escherichia coli for bioconversion of penicillins has been developed.

Polysialic acid biosynthesis and production in Escherichia coli: current state and perspectives.

Polysialic acid (PSA) is a unique polysaccharide that plays critical roles in many bioprocesses, which makes it useful in a wide range of biomedical applications. The increased demand for PSA has led to considerable efforts to improve its production using bacteria, such as Escherichia coli. Bioprocess optimization and metabolic engineering have allowed the efficient production of PSA. This review aims to summarize the metabolism of PSA with an emphasis on the importance of the key pathway components. In addition, this review provides an update on state of the art PSA production using E. coli with a special emphasis on strategies of strain engineering and process development for the enhanced production of PSA.

[Improving isoprene production by engineered heterologous mevalonate pathway in Escherichia coli].

Isoprene is an important precursor of synthetic rubber material. In our previous study, metabolic engineered Escherichia coli strain (BW-01) was constructed and used to produce isoprene. Based on the theory of protein budget, using synthetic biology strategies including the increased copy number of genes and rare codons, we regulated the expression of key enzyme to improve isoprene production in Escherichia coli strain. Under shake-flask conditions, isoprene productivity of the engineered strain (BW-07) increased by 73% compared with BW-01, reached 761.1 mg/L. It provides a reference for further studies.

Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host ß-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

Enhanced production of polysialic acid by metabolic engineering of Escherichia coli.

A number of reports have described the production of polysialic acid (PSA), focusing on the fermentation and purification of PSA. However, little work has been done to strengthen the synthetic pathway of PSA to improve PSA production. In this study, an efficient process for enhanced production of PSA using a recombinant Escherichia coli strain was developed. To improve the PSA production efficiency, the key enzymes of PSA synthetic pathway were overexpressed separately or in combination, including N-acetylneuraminate (Neu5Ac) 7-O(or 9-O)-acetyltransferase (NeuD), CMP-Neu5Ac synthetase (NeuA), and alpha-Neu5Ac alpha-2,8-sialyltransferase (NeuS). The PSA production was significantly improved by coexpression of NeuD and NeuA. In terms of the efficiency, NeuD was considered as the most important factor. Secondly, the competing pathway of intermediate Neu5Ac was blocked by nanA deletion. The efficient PSA-producing strain E. coli SA9ΔnanA/pDB1S-DA was constructed, and 16.15 ± 1.45 g/L PSA was obtained in the fed-batch culture. The production of PSA by engineered strain was increased by 85 % compared to the original strain. These results provide evidence for improvement of PSA production by regulation of the PSA biosynthetic pathway. The high productivity of our process should make it a promising cost-effective resource for PSA.

[Optimization of whole-cell biocatalysis for phenylacetyl- 7-aminodeacetoxycephalosporanic acid production].

Cephalosporins are widely used antibiotics owing to their broad activity spectra and low toxicity. Many of these medically important compounds are made chemically from 7-aminodeacetoxycephalosporanic acid. At present, this intermediate is made by synthetic ring-expansion of the inexpensive penicillin G to form G-7-ADCA, followed by enzymatic removal of the side chain to obtain 7-ADCA. The chemical synthetic process is expensive, complicated and environmentally unfriendly. Environmentally compatible enzymatic process is favorable compared with chemical synthesis. In our previous research, metabolic engineered Escherichia coli strain (H7/PG15) was constructed and used as whole-cell biocatalyst for the production of G-7-ADC with penicillin G as substrate. The whole-cell biocatalysis was studied by single factor experiment, including the composition of substrates and the conversion conditions (OD600, pH, concentration of penicillin G, MOPS, glucose, time and FeSO4). After optimization, 15 mmol/L of G-7-ADCA was obtained. The process is convenient, efficient and economic. This work would facilitate the industrial manufacturing and further product research.