Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains

Development of natural products for therapeutic use is often hindered by limited availability of material from producing organisms. The speed at which current technologies enable the cloning, sequencing, and manipulation of secondary metabolite genes for production of novel compounds has made it impractical to optimize each new organism by conventional strain improvement procedures. We have exploited the overproduction properties of two industrial organisms—Saccharopolyspora erythraea and Streptomyces fradiae, previously improved for erythromycin and tylosin production, respectively—to enhance titers of polyketides produced by genetically modified polyketide synthases (PKSs). An efficient method for delivering large PKS expression vectors into S. erythraea was achieved by insertion of a chromosomal attachment site (attB) for φC31-based integrating vectors. For both strains, it was discovered that only the native PKS-associated promoter was capable of sustaining high polyketide titers in that strain. Expression of PKS genes cloned from wild-type organisms in the overproduction strains resulted in high polyketide titers whereas expression of the PKS gene from the S. erythraea overproducer in heterologous hosts resulted in only normal titers. This demonstrated that the overproduction characteristics are primarily due to mutations in non-PKS genes and should therefore operate on other PKSs. Expression of genetically engineered erythromycin PKS genes resulted in production of erythromycin analogs in greatly superior quantity than obtained from previously used hosts. Further development of these hosts could bypass tedious mutagenesis and screening approaches to strain improvement and expedite development of compounds from this valuable class of natural products.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Genetic improvement of brewer’s yeast: current state, perspectives and limits

Brewer’s yeast strain optimisation may lead to a more efficient beer production process, better final quality or healthier beer. However, brewer’s yeast genetic improvement is very challenging, especially true when it comes to lager brewer’s yeast (Saccharomyces pastorianus) which contributes to 90% of the total beer market. This yeast is a genetic hybrid and allopolyploid. While early studies applying traditional genetic approaches encountered many problems, the development of rational metabolic engineering strategies successfully introduced many desired properties into brewer’s yeast. Recently, the first genome sequence of a lager brewer’s strain became available. This has opened the door for applying advanced omics technologies and facilitating inverse metabolic engineering strategies. The latter approach takes advantage of natural diversity and aims at identifying and transferring the crucial genetic information for an interesting phenotype. In this way, strains can be optimised by introducing “natural” mutations. However, even when it comes to self-cloned strains, severe concerns about genetically modified organisms used in the food and beverage industry are still a major hurdle for any commercialisation. Therefore, research efforts will aim at developing new sophisticated screening methods for the isolation of natural mutants with the desired properties which are based on the knowledge of genotype–phenotype linkage.     

Biotechnological production of itaconic acid and its biosynthesis in <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields—dental, ophthalmic, and drug delivery—to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.     

Chemotherapy through mitochondrial apoptosis using nutritional supplements and herbs: A brief overview

There has been increased interest in the use of naturally occurring compounds with chemopreventive and chemotherapeutic effects in the treatment of cancers. This review summarizes the most recent advances that provide new insights into the molecular mechanisms underlying the apoptotic potential of nutritional supplements and herbs. Apoptosis is an essential process in the pathogenesis of cancer and its mechanisms can be subdivided into either a death receptor-dependent extrinsic pathway or an independent (mitochondrial or intrinsic) pathway. Nutritional supplements and herbs can exert their effects on such pathways separately, sequentially, or in a manner of “crosstalk” between pathways. A strong correlation between the early collapse of the mitochondrial membrane potential and apoptosis was found for most nutritional supplements and herbs that have been studied. These observations provide examples of the development of mitochondrial targeting strategies for cancer therapy.     

Replication of Venezuelan Equine Encephalomyelitis Virus In Vitro: II. Viral Growth Response to Selected Nutritional Additives in Suspension Cultures

An attempt was made to identify nutritional additives that influence the replication of Venezuelan equine encephalomyelitis virus in suspension cultures grown in a defined serum-free medium. Proline, serine, and choline enhanced titers of the virulent PES strain; the progeny population, however, possessed a virulence character that was somewhat different from that of the PES inocula. These nutritional supplements did not appreciably influence the titers of the attenuated 9t and 20t viral strains. When both the PES and 20t strains were employed as a mixed inoculum in culture, the presence of the latter strain appeared to interfere with the growth of the PES strain and reduced its response to the medium supplements.     

The Swedish Arctic charr breeding programme

Results and experiences from a selective breeding programme aiming at improving the performance of an Arctic charr strain in aquaculture are presented. The programme, which has been running since 1985, uses traditional quantitative genetic methods based on relatedness and trait measurements. Traits considered included growth, age at sexual maturity, flesh colour, fat content and other features. Estimates in the early phase of the programme showed promising heritabilities for most traits, e.g. growth heritabilities of 0.34–0.52, and suggested that improvement by selection was feasible. Several traits such as a high rate of early maturation and poor flesh colour that were considered problematic by the Arctic charr farming industry at the programme’s start no longer hamper farming. Considerable improvement of growth by selection, estimated at 8% per generation, has contributed to shortening the production cycle in commercial farming. Results from studies of genotype–environment interactions are presented and discussed. Poor survival of fertilised eggs is a major problem in Swedish Arctic charr farming as indicated by presented survival rates. Efforts have consistently been made to avoid inbreeding in the selected strain, and at the present seventh generation the accumulated increase in inbreeding is estimated from pedigree data to be only approximately 5%.     

“微生物-肠-脑”轴介导的宿主食欲调节

人和动物的食欲受到中枢神经系统和外周激素的协同调控。近年来，一些研究指出肠道菌群的组成与变化通过多重途径影响宿主的食欲。肠道细菌分泌和产生的大量功能性代谢物如短链脂肪酸、次级胆汁酸和氨基酸衍生物等是其发挥调控作用的重要媒介物质。此外，肠道菌群还能够影响消化系统营养感知、肠迷走神经信号投递、肠道激素分泌等，这些也都会参与食欲和进食调节。在明确细菌调控食欲的作用机制后靶向调控和重组肠道微生物可能是改善宿主食欲的一种新策略，有助于厌食症、暴食症等相关疾病的诊治。     

Metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for biotechnological production of four-carbon 1,4-dicarboxylic acids

Confronted with inescapable exhaustion of the earth’s fossil energy resources, the bio-based process to produce industrial chemicals is receiving significant interest. Biotechnological production of four-carbon 1,4-dicarboxylic acids (C4 diacids) from renewable plant biomass is a promising and attractive alternative to conventional chemistry routes. Although the C4 diacids pathway is well characterized and microorganisms able to convert biomass to these acids have been isolated and described, much still has to be done to make this process economically feasible. Metabolically engineered Escherichia coli has been developed as a biocatalyst to provide new processes for the biosynthesis of many valuable chemicals. However, E. coli does not naturally produce C4 diacids in large quantities. Rational strain development by metabolic engineering based on efficient genetic tools and detailed knowledge of metabolic pathways are crucial to successful production of these compounds. This review summarizes recent efforts and experiences devoted to metabolic engineering of the industrial model bacteria E. coli that led to efficient recombinant biocatalysts for the production of C4 diacids, including succinate, fumarate, malate, oxaloacetate, and aspartate, as well as the key limitations and challenges. Continued advancements in metabolic engineering will help to improve the titers, yields, and productivities of the C4 diacids discussed here.     

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.     

Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation

Titer improvement is a constant requirement in the fermentation industry. The traditional method of “random mutation and screening” has been very effective despite the considerable amount of time and resources it demands. Rational metabolic engineering, with the use of recombinant DNA technology, provides a novel, alternative strategy for titer improvement that complements the empirical method used in industry. Manipulation of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this review, we use examples from Streptomyces secondary metabolism, the most prolific source of clinically used drugs, to demonstrate the power and utility of exploiting natural regulatory networks, in particular pathway-specific regulators, for titer improvement. Efforts to improve the titers of fredericamycin, C-1027, platensimycin, and platencin in our lab are highlighted.     

Metabolic regulation of fermentation processes

To compete in nature against other forms of life, microorganisms possess regulatory mechanisms which control production of their metabolites, thus, protecting against overproduction and excretion of these primary and secondary metabolites into the environment. To effect such an economical form of life, they possess regulatory mechanisms which control production of these metabolites and protect against overproduction and excretion into the environment of excess concentrations. In the field of industrial fermentation, the opposite concept prevails. Fermentation microbiologists search for a rare overproducing strain in nature, then further deregulate the microorganism so that it overproduces huge quantities of a desired commercially important product such as a metabolite or an enzyme. Deregulation is brought about by nutritional as well as classical and molecular genetic manipulations to bypass and/or remove negative regulatory mechanisms and to enhance positive regulatory mechanisms. These mechanisms include induction, nutritional regulation by sources of carbon, nitrogen and phosphorus, and feedback control. The controls and their modification by biotechnologists are the subjects of this review.     

Criteria for lactic acid bacteria screening to enhance silage quality

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.     

Nutritional status of the Indian population

High prevalence of low birth weight, high morbidity and mortality in children and poor maternal nutrition of the mother continue to be major nutritional concerns in India. Although nationwide intervention programmes are in operation over two decades, the situation has not changed greatly. In addition, the Indian population is passing through a nutritional transition and is expected to witness higher prevalences of adult non-communicable diseases such as diabetes, hypertension and coronary heart disease according to the theory of ‘fetal origin of adult disease’. Clearly, there is a need for examining several issues of nutritional significance for effective planning of interventions. In particular, maternal nutrition and fetal growth relationship, long term effects of early life undernutrition, interactions of prenatal nutritional experiences and postnatal undernutrition are some of the major issues that have been discussed in the present paper with the help of prospective data from various community nutrition studies carried out in the department.     

Biotechnology for Enhanced Nutritional Quality in Plants

With almost 870 million people estimated to suffer from chronic hunger worldwide, undernourishment represents a major problem that severely affects people in developing countries. In addition to undernourishment, micronutrient deficiency alone can be a cause of serious illness and death. Large portions of the world population rely on a single, starch-rich crop as their primary energy source and these staple crops are generally not rich sources of micronutrients. As a result, physical and mental health problems related to micronutrient deficiencies are estimated to affect around two billion people worldwide. The situation is expected to get worse in parallel with the expanding world population. Improving the nutritional quality of staple crops seems to be an effective and straightforward solution to the problem. Conventional breeding has long been employed for this purpose but success has been limited to the existing diversity in the gene pool. However, biotechnology enables addition or improvement of any nutrient, even those that are scarce or totally absent in a crop species. In addition, biotechnology introduces speed to the biofortification process compared to conventional breeding. Genetic engineering was successfully employed to improve a wide variety of nutritional traits over the last decade. In the present review, progress toward engineering various types of major and minor constituents for the improvement of plant nutritional quality is discussed.     

Genetic improvement of processes yielding microbial products

Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology.     

Study on the production of 6-pentyl-α-pyrone using two methods of fermentation

 The lactone 6-pentyl-α-pyrone has a characteristic coconut aroma and is produced by Trichoderma species. A study on the fermentative production of 6-pentyl-α-pyrone in both surface and submerged conditions by Trichoderma harzianum was carried out. Maximum concentrations of 455 mg/l and 167 mg/l after 96 h and 48 h of fermentation in surface and submerged conditions, respectively, were obtained without using any additional recovery operations. The resultant yields are higher than those previously reported in the literature, which may be attributable to strain characteristics in combination with the choice of fermentation conditions employed in the present study. Enough scope exists for further improvement in the yields by optimizing the cultural and nutritional parameters. Received: 13 July 1999 / Received revision: 19 November 1999 / Accepted: 19 November 1999