Omega-3 production by fermentation of Yarrowia lipolytica: From fed-batch to continuous.

The omega-3 fatty acid, cis-5,8,11,14,17-eicosapentaenoic acid (C20:5; EPA) has wide-ranging benefits in improving heart health, immune function, and mental health. A sustainable source of EPA production through fermentation of metabolically engineered Yarrowia lipolytica has been developed. In this paper, key fed-batch fermentation conditions were identified to achieve 25% EPA in the yeast biomass, which is so far the highest EPA titer reported in the literature. Dynamic models of the EPA fermentation process were established for analyzing, optimizing, and scaling up the fermentation process. In addition, model simulations were used to develop a two-stage continuous process and compare to single-stage continuous and fed- batch processes. The two stage continuous process, which is equipped with a smaller growth fermentor (Stage 1) and a larger production fermentor (Stage 2), was found to be a superior process to achieve high titer, rate, and yield of EPA. A two-stage continuous fermentation experiment with Y. lipolytica strain Z7334 was designed using the model simulation and then tested in a 2 L and 5 L fermentation system for 1,008 h. Compared with the standard 2 L fed-batch process, the two-stage continuous fermentation process improved the overall EPA productivity by 80% and EPA concentration in the fermenter by 40% while achieving comparable EPA titer in biomass and similar conversion yield from glucose. During the long-term experiment it was also found that the Y. lipolytica strain evolved to reduce byproduct and increase lipid production. This is one of the few continuous fermentation examples that demonstrated improved productivity and concentration of a final product with similar conversion yield compared with a fed-batch process. This paper suggests the two-stage continuous fermentation could be an effective process to achieve improved production of omega-3 and other fermentation products where non-growth or partially growth associated kinetics characterize the process. Biotechnol. Bioeng. 2017;114: 798-812. © 2016 Wiley Periodicals, Inc.

Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica.

The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

Construction and utilization of carotenoid reporter systems: identification of chromosomal integration sites that support suitable expression of biosynthetic genes and pathways.

In order to metabolically engineer microorganisms to produce compounds of interest, it is often desirable to integrate foreign genes into the chromosome of the host. However, the consequences of these genetic alterations are not always predictable. The use of a reporter system can often assist in determining chromosomal locations for optimal expression of foreign biosynthetic genes. The method described here involves the construction and utilization of promoterless carotenoid transposons, which provides a colorimetric screen for identifying the best chromosomal integration sites for the expression of the genes of interest. The transposons (pUTmTn5::392W and pUTmTn5::392) contain the carotenoid genes required for the production of canthaxanthin and astaxanthin, respectively. Thus, when promoterless transposons insert into the host's genome, the color of the colonies will vary based on their chromosomal location. There is a correlation between the color intensity of the colonies and the expression of the carotenoid transposon. The transposon insertion site can be determined via direct chromosomal sequencing. This sequence information is used to guide the site-specific integration of biosynthetic genes and pathways of interest.

Bioengineering of oleaginous yeast Yarrowia lipolytica for lycopene production.

Oleaginous yeast Yarrowia lipolytica is capable of accumulating large amount of lipids. There is a growing interest to engineer this organism to produce lipid-derived compounds for a variety of applications. In addition, biosynthesis of value-added products such as carotenoid and its derivatives have been explored. In this chapter, we describe methods to integrate genes involved in lycopene biosynthesis in Yarrowia. Each bacterial gene involved in lycopene biosynthesis, crtE, crtB, and crtI, will be assembled with yeast promoters and terminators and subsequently transformed into Yarrowia through random integration. The engineered strain can produce lycopene under lipid accumulation conditions.

Isolation of a Δ5 desaturase gene from Euglena gracilis and functional dissection of its HPGG and HDASH motifs.

Delta (Δ) 5 desaturase is a key enzyme for the biosynthesis of health-beneficial long chain polyunsaturated fatty acids such as arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3) via the "desaturation and elongation" pathways. A full length Δ5 desaturase gene from Euglena gracilis (EgΔ5D) was isolated by cloning the products of polymerase chain reaction with degenerate oligonucleotides as primers, followed by 5' and 3' rapid amplification of cDNA ends. The whole coding region of EgΔ5D was 1,350 nucleotides in length and encoded a polypeptide of 449 amino acids. BlastP search showed that EgΔ5D has about 39 % identity with a Δ5 desaturase of Phaeodactylum tricornutum. In a genetically modified dihomo-gamma-linoleic acid (DGLA, C20:3n-6) producing Yarrowia lipolytica strain, EgΔ5D had strong Δ5 desaturase activity with DGLA to ARA conversion of more than 24 %. Functional dissection of its HPGG and HDASH motifs demonstrated that both motifs were important, but not necessary in the exact form as encoded for the enzyme activity of EgΔ5D. A double mutant EgΔ5D-34G158G with altered sequences within both HPGG and HDASH motifs was generated and exhibited Δ5 desaturase activity similar to the wild type EgΔ5D. Codon optimization of the N-terminal region of EgΔ5D-34G158G and substitution of the arginine with serine at residue 347 improved substrate conversion to 27.6 %.

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp.

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1-crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.

Use of transposon promoter-probe vectors in the metabolic engineering of the obligate methanotroph Methylomonas sp. strain 16a for enhanced C40 carotenoid synthesis.

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.

Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a.

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the beta-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g(-1) of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.

Characterization of the Escherichia coli AaeAB efflux pump: a metabolic relief valve?

Treatment of Escherichia coli with p-hydroxybenzoic acid (pHBA) resulted in upregulation of yhcP, encoding a protein of the putative efflux protein family. Also upregulated were the adjacent genes yhcQ, encoding a protein of the membrane fusion protein family, and yhcR, encoding a small protein without a known or suggested function. The function of the upstream, divergently transcribed gene yhcS, encoding a regulatory protein of the LysR family, in regulating expression of yhcRQP was shown. Furthermore, it was demonstrated that several aromatic carboxylic acid compounds serve as inducers of yhcRQP expression. The efflux function encoded by yhcP was proven by the hypersensitivity to pHBA of a yhcP mutant strain. A yhcS mutant strain was also hypersensitive to pHBA. Expression of yhcQ and yhcP was necessary and sufficient for suppression of the pHBA hypersensitivity of the yhcS mutant. Only a few aromatic carboxylic acids of hundreds of diverse compounds tested were defined as substrates of the YhcQP efflux pump. Thus, we propose renaming yhcS, yhcR, yhcQ, and yhcP, to reflect their role in aromatic carboxylic acid efflux, to aaeR, aaeX, aaeA, and aaeB, respectively. The role of pHBA in normal E. coli metabolism and the highly regulated expression of the AaeAB efflux system suggests that the physiological role may be as a "metabolic relief valve" to alleviate toxic effects of imbalanced metabolism.