Combining evolutionary and metabolic engineering in Rhodosporidium toruloides for lipid production with non-detoxified wheat straw hydrolysates.

Improving the yield of carbohydrate to lipid conversion and lipid productivity are two critical goals to develop an economically feasible process to commercialize microbial oils. Lignocellulosic sugars are potential low-cost carbon sources for this process but their use is limited by the toxic compounds produced during biomass pretreatment at high solids loading, and by the pentose sugars (mainly xylose) which are not efficiently metabolized by many microorganisms. Adaptive laboratory evolution was used to select a Rhodosporidium toruloides strain with robust growth in non-detoxified wheat straw hydrolysates, produced at 20% solids loading, and better xylose consumption rate. An arabinose-inducible cre-lox recombination system was developed in this evolved strain that was further engineered to express a second copy of the native DGAT1 and SCD1 genes under control of the native xylose reductase (XYL1) promoter. Fed-batch cultivation of the engineered strain in 7-L bioreactors produced 39.5 g lipid/L at a rate of 0.334 g/Lh-1 and 0.179 g/g yield, the best results reported in R. toruloides with non-detoxified lignocellulosic hydrolysates to date.

Oleaginous yeasts: Promising platforms for the production of oleochemicals and biofuels.

Oleaginous yeasts have a unique physiology that makes them the best suited hosts for the production of lipids, oleochemicals, and diesel-like fuels. Their high lipogenesis, capability of growing on many different carbon sources (including lignocellulosic sugars), easy large-scale cultivation, and an increasing number of genetic tools are some of the advantages that have encouraged their use to develop sustainable processes. This mini-review summarizes the metabolic engineering strategies developed in oleaginous yeasts within the last 2 years to improve process metrics (titer, yield, and productivity) for the production of lipids, free fatty acids, fatty acid-based chemicals (e.g., fatty alcohols, fatty acid ethyl esters), and alkanes. During this short period of time, tremendous progress has been made in Yarrowia lipolytica, the model oleaginous yeast, which has been engineered to improve lipid production by different strategies including increasing lipogenic pathway flux and biosynthetic precursors, and blocking degradation pathways. Moreover, remarkable advances have also been reported in Rhodosporidium toruloides and Lipomyces starkey despite the limited genetic tools available for these two very promising hosts. Biotechnol. Bioeng. 2017;114: 1915-1920. © 2017 Wiley Periodicals, Inc.

Engineering Rhodosporidium toruloides for the production of very long-chain monounsaturated fatty acid-rich oils.

Erucic acid (cis-docosa-13-enoic acid, C22:1∆13) and nervonic acid (cis-tetracosa-15-enoic acid, C24:1 ∆15) are important renewable feedstocks in plastic, cosmetic, nylon, and lubricant industries. Furthermore, nervonic acid is also applied to the treatment of some neurological diseases. However, the production of these two very long-chain fatty acids (VLCFA) is very limited as both are not present in the main vegetable oils (e.g., soybean, rapeseed, sunflower, and palm). Ectopic integration and heterologous expression of fatty acid elongases (3-ketoacyl-CoA synthases, KCS) from different plants in Rhodosporidium toruloides resulted in the de novo synthesis of erucic acid and nervonic acid in this oleaginous yeast. Increasing KCS gene copy number or the use of a push/pull strategy based on the expression of elongases with complementary substrate preferences increased significantly the amount of these two fatty acids in the microbial oils. Oil titers in 7-L bioreactors were above 50 g/L, and these two VLCFA represented 20-30% of the total fatty acids. This is the first time that microbial production of these types of oils is reported.

Microbial production of fatty alcohols.

Fatty alcohols have numerous commercial applications, including their use as lubricants, surfactants, solvents, emulsifiers, plasticizers, emollients, thickeners, and even fuels. Fatty alcohols are currently produced by catalytic hydrogenation of fatty acids from plant oils or animal fats. Microbial production of fatty alcohols may be a more direct and environmentally-friendly strategy since production is carried out by heterologous enzymes, called fatty acyl-CoA reductases, able to reduce different acyl-CoA molecules to their corresponding primary alcohols. Successful examples of metabolic engineering have been reported in Saccharomyces cerevisiae and Escherichia coli in which the production of fatty alcohols ranged from 1.2 to 1.9 g/L, respectively. Due to their metabolic advantages, oleaginous yeasts are considered the best hosts for production of fatty acid-derived chemicals. Some of these species can naturally produce, under specific growth conditions, lipids at high titers (>50 g/L) and therefore provide large amounts of fatty acyl-CoAs or fatty acids as precursors. Very recently, taking advantage of such features, over 8 g/L of C16-C18 fatty alcohols have been produced in Rhodosporidium toruloides. In this review we summarize the different metabolic engineering strategies, hosts and cultivation conditions used to date. We also point out some future trends and challenges for the microbial production of fatty alcohols.

Fatty alcohols production by oleaginous yeast.

We have engineered Rhodosporidium toruloides to produce fatty alcohols by expressing a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8. Production of fatty alcohols in flasks was achieved in different fermentation media at titers ranging from 0.2 to 2 g/L. In many of the conditions tested, more than 80 % of fatty alcohols were secreted into the cultivation broth. Through fed-batch fermentation in 7 L bioreactors, over 8 g/L of C(16)-C(18) fatty alcohols were produced using sucrose as the substrate. This is the highest titer ever reported on microbial production of fatty alcohols to date.

Contributions of microorganisms to industrial biology.

Life on earth is not possible without microorganisms. Microbes have contributed to industrial science for over 100 years. They have given us diversity in enzymatic content and metabolic pathways. The advent of recombinant DNA brought many changes to industrial microbiology. New expression systems have been developed, biosynthetic pathways have been modified by metabolic engineering to give new metabolites, and directed evolution has provided enzymes with modified selectability, improved catalytic activity and stability. More and more genomes of industrial microorganisms are being sequenced giving valuable information about the genetic and enzymatic makeup of these valuable forms of life. Major tools such as functional genomics, proteomics, and metabolomics are being exploited for the discovery of new valuable small molecules for medicine and enzymes for catalysis.

Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation.

Microbes have been good to us. They have given us thousands of valuable products with novel structures and activities. In nature, they only produce tiny amounts of these secondary metabolic products as a matter of survival. Thus, these metabolites are not overproduced in nature, but they must be overproduced in the pharmaceutical industry. Genetic manipulations are used in industry to obtain strains that produce hundreds or thousands of times more than that produced by the originally isolated strain. These strain improvement programs traditionally employ mutagenesis followed by screening or selection; this is known as 'brute-force' technology. Today, they are supplemented by modern strategic technologies developed via advances in molecular biology, recombinant DNA technology, and genetics. The progress in strain improvement has increased fermentation productivity and decreased costs tremendously. These genetic programs also serve other goals such as the elimination of undesirable products or analogs, discovery of new antibiotics, and deciphering of biosynthetic pathways.

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.

Antifungals.

The need for new antifungal agents is undeniable. Current therapeutic choices for the treatment of invasive fungal infections are limited to three classes of drugs. Most used antifungal agents are not completely effective due to the development of resistance, host toxicity and undesirable side effects that limit their use in medical practice. Invasive fungal infections have significantly increased over the last decades and the mortality rates remain unacceptably high. More threatening, new resistance patterns have been observed including simultaneous resistance to different antifungal classes. In the last years, deeper insights into the molecular mechanisms for fungal resistance and virulence have yielded some new potential targets for antifungal therapeutics. Chemical genomics-based screenings, high throughput screenings of natural products and repurposing of approved drugs are some of the approaches being followed for the discovery of new antifungal molecules. However, despite the emerging need for effective antifungal agents, the current pipeline contains only a few promising molecules, with novel modes of action, in early clinical development stages.

Expression of an Aspergillus niger glucose oxidase in saccharomyces cerevisiae and its use to optimize fructo-oligosaccharides synthesis.

Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.

Microbial enzymes: tools for biotechnological processes.

Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

Essential role of genetics in the advancement of biotechnology.

Microorganisms are one of the greatest sources of metabolic and enzymatic diversity. In recent years, emerging recombinant DNA and genomic techniques have facilitated the development of new efficient expression systems, modification of biosynthetic pathways leading to new metabolites by metabolic engineering, and enhancement of catalytic properties of enzymes by directed evolution. Complete sequencing of industrially important microbial genomes is taking place very rapidly and there are already hundreds of genomes sequenced. Functional genomics and proteomics are major tools used in the search for new molecules and development of higher-producing strains.

Semisynthesis of novel monacolin J derivatives: hypocholesterolemic and neuroprotective activities.

A fungal strain able to naturally accumulate large amounts of monacolin J was improved by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis and genetic disruption of the lovF gene. Semisynthesis was then used to produce novel statins by attaching different side chains at the C8 hydroxyl residue. In vitro hypocholesterolemic and neuroprotection assays showed that one derivative (NST0037) had a very low 3-hydroxy-3-methylglutaryl CoA reductase IC(50) and high protection rate for oxidative-stress-induced neuron cell death.

Metabolism of prebiotic products containing beta(2-1) fructan mixtures by two Lactobacillus strains.

The capacity of two probiotic strains, isolated from human breast milk, to use several beta(2-1) fructan mixtures as carbon and energy source in in vitro cultures has been tested. Results showed that both strains, Lactobacillus gasseri CECT5714 and Lactobacillus fermentum CECT5716, reached higher growth levels on culture media containing fructooligosaccharide mixtures produced by enzymatic synthesis, compared to those obtained by inulin hydrolysis. Furthermore, the shortest beta(2-1) fructan, kestose, was the only prebiotic compound in the mixtures significantly metabolized in all growth media tested. Analysis of short-chain fatty acid production showed no correlation between the fatty acid profile produced and the carbon source used in each experiment. These data could serve to select appropriate beta(2-1) fructans to be used as prebiotics for L. gasseri CECT5714 and L. fermentum CECT5716 and to design suitable symbiotic food products containing the mentioned lactobacilli.

High-level expression and characterization of Galactomyces geotrichum (BT107) lipase I in Pichia pastoris.

The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.

Fungal biotechnology.

Fungi are used in many industrial processes, such as the production of enzymes, vitamins, polysaccharides, polyhydric alcohols, pigments, lipids, and glycolipids. Some of these products are produced commercially while others are potentially valuable in biotechnology. Fungal secondary metabolites are extremely important to our health and nutrition and have tremendous economic impact. In addition to the multiple reaction sequences of fermentations, fungi are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine-chemical industry in the production of single-isomer intermediates. Recombinant DNA technology, which includes yeasts and other fungi as hosts, has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as a means of increasing titers and yields of microbial processes and in the discovery of new drugs. Today, fungal biology is a major participant in global industry. Moreover, the best is yet to come as genomes of additional species are sequenced at some level (cDNA, complete genomes, expressed sequence tags) and gene and protein arrays become available.