Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli

The heterologous biosynthesis of complex polyketides in Escherichia coli was recently achieved through metabolic engineering. However, it was observed that less than 10% of the propionate carbon source is transformed into the erythromycin precursor, 6-deoxyerythronolide B (6dEB), resulting in a 1.4% molar yield. Therefore, metabolic flux analysis was performed using a model of the Escherichia coli metabolism with the addition of the enzymes required for 6dEB synthesis. The analysis shows that the maximum theoretical yield for 6dEB synthesis in E. coli is 11%. The maintenance energy requirement of E. coli and limitations in the specific oxygen uptake rate can further decrease the yield, suggesting that the observed 6dEB yield of 1.4% can be the result of these two factors. In addition, the results suggest that an increase in the specific carbon and oxygen uptake rates will increase the yield of 6dEB. The use of glucose as an alternative carbon source was also evaluated using metabolic flux analysis and the results suggest that the choice of glucose as the carbon source will allow a small improvement in performance relative to a propionate-based process.     

Improved heterologous erythromycin A production through expression plasmid re‐design

The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli‐derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species‐specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5‐fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:862–869, 2013     

利用合成调控RNA技术提高大肠杆菌异源合成红霉素前体6-脱氧红霉内酯B产量

红霉素为代表的聚酮类化合物已经成功的在大肠杆菌中实现了异源合成,但其产量仍然较低(仅~10 mg/L)。本研究基于大肠杆菌全基因组代谢模型iAF1260,利用通量平衡分析预测了红霉素母核6-脱氧红霉内酯(6-Deoxyerythronolide B,6-d EB)生物合成的关键靶点,通过合成调控RNA技术(Synthetic small regulatory RNAs,sRNAs)对预测的靶点进行验证。结果表明,以弱化lsrC(编码LsrABC转运蛋白)和ack A(编码乙酸激酶蛋白)为代表的关键靶点改造可以显著提高6-d EB异源合成,提高幅度可达48.7%。通过弱化靶点的组合,进一步改善了6-d EB的异源合成,产量最终可达22.8 mg/L,比出发菌株产量提高59.9%。本研究发现和确认了6个有效的调控靶点,最终成功地改善了6-d EB在大肠杆菌中的异源合成。研究表明,通量分布比较分析结合sRNAs技术是一种有效的方法提高6-d EB异源合成,也为改善其他代谢产物的异源合成提供了可供借鉴的研究思路。     

Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

Expression of key glycosphingolipid biosynthesis‐globo series pathway genes in Escherichia coli F18‐resistant and Escherichia coli F18‐sensitive piglets

A pioneering study showed that the glycosphingolipid biosynthesis‐globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1 and NAGA) may play an important regulatory role in resistance to Escherichia coli F18 in piglets. Therefore, we analysed differential gene expression in 11 tissues of two populations of piglets sensitive and resistant respectively to E. coli F18 and the correlation of differential gene expression in duodenal and jejunal tissues. We found that the mRNA expression of the seven genes was relatively high in spleen, liver, lung, kidney, stomach and intestinal tract; the levels in thymus and lymph nodes were lower, with the lowest levels in heart and muscle. FUT2 gene expression in the duodenum and jejunum of the resistant population was significantly lower than that in the sensitive group (P < 0.01). ST3GAL1 gene expression was also significantly lower in the duodenum of the resistant population than in the sensitive group (P < 0.05). No significant differences were observed among the remaining genes. The expression level of FUT1 was extremely significantly positively correlated with FUT2 and B3GALNT1 expression (P < 0.01) and also had a significant positive correlation with NAGA expression (P < 0.05). The expression level of FUT2 had extremely significant positive correlations with FUT1, ST3GAL1 and B3GALNT1 (P < 0.01). These results suggest that FUT2 plays an important role in E. coli F18 resistance in piglets. FUT1, ST3GAL1, B3GALNT1 and NAGA may also participate in the mechanism of resistance to E. coli F18.     

Constructing E. coli Co‐Cultures for De Novo Biosynthesis of Natural Product Acacetin

Modular co‐culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co‐cultures composed of two and three Escherichia coli strains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value‐added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicated E. coli strain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co‐cultures show a 4.83‐fold improvement in production comparing to the mono‐culture controls. Importantly, cultivation of the three‐strain co‐culture in shake flasks result in the production of 20.3 mg L^?1 acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co‐culture engineering for complex flavonoid biosynthesis.     

New tools for recombinant protein production in Escherichia coli: A 5‐year update

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost‐effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.     

Improved heterologous production of the nonribosomal peptide‐polyketide siderophore yersiniabactin through metabolic engineering and induction optimization

Biosynthesis of complex natural products like polyketides and nonribosomal peptides using Escherichia coli as a heterologous host provides an opportunity to access these molecules. The value in doing so stems from the fact that many compounds hold some therapeutic or other beneficial property and their original production hosts are intractable for a variety of reasons. In this work, metabolic engineering and induction variable optimization were used to increase production of the polyketide‐nonribosomal peptide compound yersiniabactin, a siderophore that has been utilized to selectively remove metals from various solid and aqueous samples. Specifically, several precursor substrate support pathways were altered through gene expression and exogenous supplementation in order to boost production of the final compound. The gene expression induction process was also analyzed to identify the temperatures and inducer concentrations resulting in highest final production levels. When combined, yersiniabactin production was extended to ～175 mg L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1412–1417, 2016     

Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

In the genome‐engineering era, it is increasingly important that researchers have access to a common set of platform strains that can serve as debugged production chassis and the basis for applying new metabolic engineering strategies for modeling and characterizing flux, engineering complex traits, and optimizing overall performance. Here, we describe such a platform strain of E. coli engineered for ethanol production. Starting with a fully characterized host strain (BW25113), we site‐specifically integrated the genes required for homoethanol production under the control of a strong inducible promoter into the genome and deleted the genes encoding four enzymes from competing pathways. This strain is capable of producing >30 g/L of ethanol in minimal media with <2 g/L produced of any fermentative byproduct. Using this platform strain, we tested previously identified ethanol tolerance genes and found that while tolerance was improved under certain conditions, any effect on ethanol production or tolerance was lost when grown under production conditions. Thus, our findings reinforce the need for a metabolic engineering “commons” that could provide a set of platform strains for use in more sophisticated genome‐engineering strategies. Towards this end, we have made this production strain available to the scientific community. Biotechnol. Bioeng. 2013; 110: 1520–1526. © 2013 Wiley Periodicals, Inc.     

False‐negative β‐d‐glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms and Escherichia coli from water

Aims: Testing for β‐d ‐glucuronidase activity has become the basis of many methods for the detection of Escherichia coli in both food and water. Used in combination with tests for the presence of β‐d ‐glucuronidase, these tests offer a simple method for simultaneously detecting coliforms and E. coli. The purpose of this study was to determine the effectiveness of several different procedures in detecting β‐d ‐glucuronidase activity and hence in detecting E. coli. Methods and Results: The ability of membrane lactose glucuronide agar (MLGA), Colilert‐18^®, MI agar, Colitag^® and Chromocult agar to detect β‐d ‐glucuronidase activity was tested with over 1000 isolates of E. coli recovered from naturally contaminated water samples. Four of the media gave very similar results but MLGA failed to detect glucuronidase activity in 15·6% of the cultures tested. Conclusions: MLGA had very poor sensitivity for the detection of β‐d ‐glucuronidase activity in strains of E. coli isolated from naturally contaminated water. This is probably because of the fact that β‐d ‐glucuronidase activity is pH‐sensitive and that acid is formed by E. coli during fermentation of lactose in MLGA. Significance and Impact of the Study: The detection of E. coli in drinking water is the primary test used to establish faecal contamination. The poor sensitivity of MLGA in detecting β‐d ‐glucuronidase activity suggests that this medium and others containing high concentrations of fermentable carbohydrate should not be used for the detection of E. coli.     

Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-acyl carrier protein transacylase

The microbial biosynthesis of free fatty acid, which can be used as precursors for the production of fuels or chemicals from renewable carbon sources, has attracted significant attention in recent years. Free fatty acids can be produced by introducing an acyl-carrier protein (ACP) thioesterase (TE) gene into Escherichia coli. The first committed step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl-CoA by an adenosine triphosphate (ATP)-dependent acetyl-CoA carboxylase followed by the conversion of malonyl-CoA to malonyl-ACP through the enzyme malonyl CoA-acyl carrier protein transacylase (MCT; FabD). The E. coli fabD gene encoding MCT has been cloned and studied. However, the effect of FabD overexpression in a fatty acid overproducing strain has not been examined. In this study, we examined the effect of FabD overexpression in a fatty acid overproducing strain carrying an acyl-ACP TE. Specifically, the effect of overexpressing a fabD gene from four different organisms on fatty acid production was compared. The strains carrying a fabD gene from E. coli, Streptomyces avermitilis MA-4680, or Streptomyces coelicolor A3(2) improved the free fatty acid production; these three strains produced more free fatty acids, about 11% more, than the control strain. The strain carrying a fabD gene from Clostridium acetobutylicum ATCC 824, however, produced similar quantities of free fatty acids as the control strain. In addition, the three FabD overexpressed strains also have higher fatty acid/glucose yields. The results suggested that FabD overexpression can be used to improve free fatty acid production by increasing the malonyl-ACP availability.