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.     

Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and β-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.     

Minimal polyketide pathway expression in an actinorhodin cluster-deleted and regulation-stimulated <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Along with traditional random mutagenesis-driven strain improvement, cloning and heterologous expression of Streptomyces secondary metabolite gene clusters have become an attractive complementary approach to increase its production titer, of which regulation is typically under tight control via complex multiple regulatory networks present in a metabolite low-producing wild-type strain. In this study, we generated a polyketide non-producing strain by deleting the entire actinorhodin cluster from the chromosome of a previously generated S. coelicolor mutant strain, which was shown to stimulate actinorhodin biosynthesis through deletion of two antibiotic downregulators as well as a polyketide precursor flux downregulator (Kim et al. in Appl Environ Microbiol 77:1872–1877, 2011). Using this engineered S. coelicolor mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product, was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis. Aloesaponarin II production was detected only in the presence of a pathway-specific regulatory gene, actII-ORF4, and its production level was the highest in the actinorhodin cluster-deleted and downregulator-deleted mutant strain, implying that this engineered polyketide pathway-free and regulation-optimized S. coelicolor mutant strain could be used as a general surrogate host for efficient expression of indigenous or foreign polyketide pathways derived from diverse actinomycetes in nature.     

Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA,B, C subunits

The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.     

Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d ‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.     

Synthetic sRNA‐Based Engineering of Escherichia coli for Enhanced Production of Full‐Length Immunoglobulin G

Production of monoclonal antibodies (mAbs) receives considerable attention in the pharmaceutical industry. There has been an increasing interest in the expression of mAbs in Escherichia coli for analytical and therapeutic applications in recent years. Here, a modular synthetic biology approach is developed to rationally engineer E. coli by designing three functional modules to facilitate high‐titer production of immunoglobulin G (IgG). First, a bicistronic expression system is constructed and the expression of the key genes in the pyruvate metabolism is tuned by the technologies of synthetic sRNA translational repression and gene overexpression, thus enhancing the cellular material and energy metabolism of E. coli for IgG biosynthesis (module 1). Second, to prevent the IgG biodegradation by proteases, the expression of a number of key proteases is identified and inhibited via synthetic sRNAs (module 2). Third, molecular chaperones are co‐expressed to promote the secretion and folding of IgG (module 3). Synergistic integration of the three modules into the resulting recombinant E. coli results in a yield of the full‐length IgG ≈150 mg L^?1 in a 5L fed‐batch bioreactor. The modular synthetic biology approach could be of general use in the production of recombinant mAbs.     

Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15°C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.     

Metabolic engineering of<Emphasis Type="Italic"> Escherichia coli</Emphasis> for improved 6-deoxyerythronolide B production

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit (2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase (matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of ¹³C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.S. Murli and J. Kennedy contributed equally to this work     

A two-vector system for the production of recombinant polyketides in Streptomyces

A two-vector system was developed for heterologous expression of the three genes comprising the 6-deoxyerythronolide B synthase (DEBS) polyketide gene cluster. Individual DEBS genes and pairwise combinations of two such genes were each cloned downstream of the actinorhodin (actI) promoter in two compatible Streptomyces vectors: the autonomously replicating vector, pKAO127′Kan′, and the integrating vector, pSET152. The resulting plasmids were either simultaneously or sequentially transformed into Streptomyces lividans K4-114. Efficient trans-complementation of modular polyketide synthase subunit proteins occurred when the respective genes were transcribed from the two vectors and resulted in production of the erythromycin precursor 6-deoxyerythronolide B (6-dEB). Journal of Industrial Microbiology & Biotechnology (2000) 24, 46–50. Received 17 March 1999/ Accepted in revised form 15 September 1999     

Improved phloroglucinol production by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.     

Recombinatorial biosynthesis of polyketides

Modular polyketide synthases (PKSs) from Streptomyces and related genera of bacteria produce many important pharmaceuticals. A program called CompGen was developed to carry out in silico homologous recombination between gene clusters encoding PKSs and determine whether recombinants have cluster architectures compatible with the production of polyketides. The chemical structure of recombinant polyketides was also predicted. In silico recombination was carried out for 47 well-characterised clusters. The predicted recombinants would produce 11,796 different polyketide structures. The molecular weights and average degree of reduction of the chemical structures are dispersed around the parental structures indicating that they are likely to include pharmaceutically interesting compounds. The details of the recombinants and the chemical structures were entered in a database called r-CSDB. The virtual compound library is a useful resource for computer-aided drug design and chemoinformatics strategies for finding pharmaceutically relevant chemical entities. A strategy to construct recombinant Streptomyces strains to produce these polyketides is described and the critical steps of mobilizing large biosynthetic clusters and producing new linear cloning vectors are illustrated by experimental data.     

Recombinant production of omega‐3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1

Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.     

Heterologous production of epothilone C and D in Escherichia coli

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.     

Target selection of soluble protein complexes for structural proteomics studies

Background  

Protein expression in E. coli is the most commonly used system to produce protein for structural studies, because it is fast and inexpensive and can produce large quantity of proteins. However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise [1]. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes. To investigate how the E. coli protein expression system could be used for this purpose, we purified apoptotic binary protein complexes formed between members of the Caspase Associated Recruitment Domain (CARD) family.     

On the functional organization of the arg ECBH cluster of genes in Escherichia coli K-12

Summary Among the four seemingly adjacent loci of the argECBH cluster of E. coli K-12, the last three are shown to belong to the same unit of coordinated expression; the latter exhibits a clockwise polarity in contrast to all other known E. coli operons, except the cluster governing the synthesis of the pyruvate dehydrogenase complex.The analysis of several deletion and nonsense mutants suggests that argE (the expression of which is not strictly correlated with the functioning of the argCBH group) has the same polarity but is not integrated with the three other genes into one operon.Between polar argC B and B mutants the coefficient of repressibility of enzyme H synthesis varies widely. This feature resembles the reduced repressibility of distal gene activity found in polar mutants in the tryptophan operons of E. coli and S. typhimurium but not in the lac, gal (E. coli) and his (S. typhimurium) operons.Possible implications of the present results and some relevant data that have appeared in the recent literature are discussed.Research fellow of the Institute for the Encouragement of Scientific Research in Industry and Agriculture, Belgium.Research fellow of the National Fund of Scientific Research, Belgium.     

Characterization of E. coli O24 and O56 O Antigen Gene Clusters Reveals a Complex Evolutionary History of the O24 Gene Cluster

O antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria. It has many different forms, which are almost entirely due to genetic variations of O antigen gene clusters. In this study, the O antigen gene clusters of E. coli O24 and O56 were sequenced, and all genes were assigned functions on the basis of homology. Comparison of O antigen gene clusters indicated that E. coli O24 O antigen gene cluster has possibly arisen from the E. coli O56 gene cluster, through inactivation of two glycosyltransferase genes and acquisition of two new genes from E. coli O157 and O152, respectively. The insertion sequence elements seemed to play important roles for the assembly of the O24 O antigen gene cluster. This is the first time that the evolutionary history of a multi-origin O antigen gene cluster is clearly demonstrated. Genes specific to E. coli O24 and O56 were also identified, which may be used for development of DNA-based serotyping schemes.     

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation—up to 6 mM—in the medium. This yield is 21.5-fold higher than that obtained in previous studies.     

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.