genetic-engineering-an-unconventional-yeast-for-renewable-biofuel

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

National University of Singapore

A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-γ-linolenic acid (20:3 Δ(8,11,14)), resulting in arachidonic acid (20:4 Δ(5,8,11,14)) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5'-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5'-upstream region of other delta5 desaturases showed that the 5'-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5'-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5'-upstream region of fad5 could drive the expression of green fluorescent protein.

Cloning and functional identification of delta5 fatty acid desaturase gene and its 5'-upstream region from marine fungus Thraustochytrium sp. FJN-10.

On the molecular and biochemical levels, the effects of different carbon sources on biomass production, fatty acid biosynthesis, and gene expression of three desaturases were investigated in Mortierella alpina ATCC 16266, at a stationary phase, which is an important filamentous fungus capable of producing various polyunsaturated fatty acids (PUFAs). The maximum mycelial biomass was achieved using sucrose as carbon source. However, the highest productivity of total lipids was shown to be no biomass associated. In addition, glucose was the preferred carbon source for the expression of three desaturase genes compared to others, but the change at the corresponding fatty acid product's level of these desaturase genes was not in accordance with the change measured at the mRNA level among those carbon sources that we utilized. Significant discrepancies between the mRNA expression and the product abundance may indicate post-transcriptional regulatory mechanisms of these desaturases.

Effects of different carbon sources on the growth, fatty acids production, and expression of three desaturase genes of Mortierella alpina ATCC 16266.

Unsaturated fatty acids (UFAs), including oleic acid (OA, C18:1n-9), linoleic acid (LA, C18:2n-6) and α-linolenic acid (ALA, C18:3n-3), are major components of membrane lipids in Pichia pastoris GS115. In order to clarify the biosynthesis pathway of UFAs on the molecular level and investigate their possible roles in growth and development of this strain, we here report modified strains with disrupted desaturase gene by homologous recombination. Gas chromatography analysis of fatty acid composition in the corresponding mutants confirmed that ∆(12)-desaturase encoded by Fad12 was responsible for the formation of LA, and ALA was synthesized by ∆(15)-desaturase encoded by Fad15. Simultaneous deletion of Fad9A and Fad9B was lethal and supplementation of OA could restore growth, indicating that possibly both Fad9A and Fad9B encoded ∆(9)-desaturase that converted SA into OA. Phenotypic analysis demonstrated that wild type and Fad15 mutant grew at almost the same rate, Fad12 mutant grew much slower than these two strains. Moreover, OA was positively correlated to cold tolerance and ethanol tolerance of GS115, whereas LA and ALA did not affect cold tolerance and ethanol tolerance of it. In addition, we showed that tolerance of GS115 to high concentration of methanol was independent of these three UFAs.

Knockout of fatty acid desaturase genes in Pichia pastoris GS115 and its effect on the fatty acid biosynthesis and physiological consequences.

Here we investigated the regulation of Pichia pastoris desaturase genes by low temperature and exogenous fatty acids in the late-exponential phase at the transcriptional level. Time-course studies of gene expression showed that mRNA levels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous oleic acid. Stearic acid showed no obvious repression of mRNA levels of Fad12 and Fad15 and a slight increase in Fad9A and Fad9B mRNA levels. Using a promoter-reporter gene construct, we demonstrated that the pFAD15 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by unsaturated fatty acids. Also, there was no absolute correlation between mRNA abundance and production of corresponding fatty acids. Disruption of Spt23 resulted in a decrease in transcript levels of Fad9A and Fad9B, but had little effect on the other desaturase genes. Consistent with these observations, a decrease in the relative amount of oleic acid (OLA) and an increase in the relative content of linoleic acid and ALA with different degrees were clearly observed in the stationary phase cells of ΔSpt23 mutant. Further analysis showed that the effect of low-temperature activation and OLA inhibition on expression of Fad9A and Fad9B seemed to disappear after disruption of the Spt23 gene, which indicated that Spt23p is essential for the expression of two Δ9-desaturase genes internally and probably involved in the regulation of Δ9-desaturase genes transcription in response to external stimuli, and thereby plays a role in the synthesis of OLA.

Transcriptional regulation of desaturase genes in Pichia pastoris GS115.

Fatty acid derivatives, such as hydroxy fatty acids, fatty alcohols, fatty acid methyl/ethyl esters, and fatty alka(e)nes, have a wide range of industrial applications including plastics, lubricants, and fuels. Currently, these chemicals are obtained mainly through chemical synthesis, which is complex and costly, and their availability from natural biological sources is extremely limited. Metabolic engineering of microorganisms has provided a platform for effective production of these valuable biochemicals. Notably, synthetic biology-based metabolic engineering strategies have been extensively applied to refactor microorganisms for improved biochemical production. Here, we reviewed: (i) the current status of metabolic engineering of microbes that produce fatty acid-derived valuable chemicals, and (ii) the recent progress of synthetic biology approaches that assist metabolic engineering, such as mRNA secondary structure engineering, sensor-regulator system, regulatable expression system, ultrasensitive input/output control system, and computer science-based design of complex gene circuits. Furthermore, key challenges and strategies were discussed. Finally, we concluded that synthetic biology provides useful metabolic engineering strategies for economically viable production of fatty acid-derived valuable chemicals in engineered microbes.

Production of Fatty Acid-derived valuable chemicals in synthetic microbes.

The mammalian peroxiredoxin (PRX) family contains six members that provide antioxidant defense in different cell types by removing reactive oxygen species (ROS) through conserved active cysteines. Different from other members, PRX3 is predominantly located in mitochondria, a major apoptosis mediator. The purpose of this review is to summarize the findings on PRX3 concerning its role in ROS removal, apoptosis, and chemoresistance of cancer cells.

The functional role of peroxiredoxin 3 in reactive oxygen species, apoptosis, and chemoresistance of cancer cells.

Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical.

Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

The human gut microbiota has a significant effect on many aspects of human physiology such as metabolism, nutrient absorption, and immune function. Imbalance of the microbiota has been implicated in many disorders including inflammatory bowel disease, obesity, asthma, psychiatric illnesses, and cancers. As a kind of functional foods, probiotics have been shown to play a protective role against cancer development in animal models. Clinical application of probiotics indicated that some probiotic strains could diminish the incidence of postoperative inflammation in cancer patients. Chemotherapy or radiotherapy-related diarrhea was relieved in patients who were administered oral probiotics. The present review summarizes the up-to-date studies on probiotic effects and the underlying mechanisms related to cancer. At present, it is commonly accepted that most commercial probiotic products are generally safe and can improve the health of the host. By modulating intestinal microbiota and immune response, some strains of probiotics can be used as an adjuvant for cancer prevention or/and treatment.

Ai-Qun Yu

National University of Singapore

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production