- NSF-PAR ID:
- 10059480
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 115
- Issue:
- 9
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- p. 2383-2388
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. UnlikeSaccharomyces cerevisiae, K. marxianus grows on low‐cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluatedK. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2‐pyrone synthase (2‐PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high‐stability 2‐PS mutant and a promoter substitution increased titer four‐fold. 2‐PS expression from a multi‐copy pKD1‐based plasmid improved TAL titers a further five‐fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed‐batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production. -
more » « less
The engineering of
Yarrowia lipolytica to accumulate lipids with high titers and productivities has been enabled with a handful of constitutive promoters for pathway engineering. However, the development of promoters that are both strong and lipid responsive could greatly benefit the bioproduction efficiency of lipid‐derived oleochemicals in oleaginous yeast. In this study, a fatty acid regulated hybrid promoter for use inY. lipolytica is engineered. A 200 bp upstream regulatory sequence in the peroxisomal acyl CoA oxidase 2 (POX2) promoter is identified. Further analysis of the promoter sequence reveal a regulatory sequence, that when used in tandem repeats, lead to a 48‐fold induction of gene expression relative to glucose and fourfold higher than the native POX2 promoter. To date, this is the strongest inducible promoter reported inY. lipolytica . Taken together, the results show that it is possible to engineer strong promoters that retain strong inducibility. These types of promoters will be useful in controlling metabolism and as fatty acid sensors. -
more » « less
Abstract The United States produces more than 10 million tons of waste oils and fats each year. This paper aims to establish a new biomanufacturing platform that converts waste oils or fats into a series of value‐added products. Our research employs the oleaginous yeast
Yarrowia lipolytica as a case study for citric acid (CA) production from waste oils. First, we conducted the computational fluid dynamics (CFD) simulation of the bioreactor system and identified that the extracellular mixing and mass transfer is the first limiting factor of an oil fermentation process due to the insolubility of oil in water. Based on the CFD simulation results, the bioreactor design and operating conditions were optimized and successfully enhanced oil uptake and bioconversion in fed‐batch fermentation experiments. After that, we investigated the impacts of cell morphology on oil uptake, intracellular lipid accumulation, and CA formation by overexpressing and deleting theMHY1 gene in the wild typeY. lipolytica ATCC20362. Fairly good linear correlations (R 2 > 0.82) were achieved between cell morphology and productivities of biomass, lipid, and CA. Finally, fermentation kinetics with both glucose and oil substrates were compared and the oil fermentation process was carefully evaluated. Our study suggests that waste oils or fats can be economical feedstocks for biomanufacturing of many high‐value products. -
more » « less
Abstract Photosynthetic microalgae like
Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids.Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate howN. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase fromDaphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g−1total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐d ‐erythritol 4‐phosphate pathway,Coleus forskohlii 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g−1DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae. -
Background: Although microalgal biofuels have potential advantages over conventional fossil fuels, high production costs limit their application in the market. We developed bio-flocculation and incubation methods for the marine alga Nannochloropsis oceanica CCMP1779 and the oleaginous fungus Mortierella elongata AG77 resulting in increased oil productivity. Results: Grown separately and then combining the cells the M. elongata mycelium could efficiently capture N. oceanica due to an intricate cellular interaction between the two species leading to bio-flocculation. Use of a high-salt culture medium induced accumulation of triacylglycerol (TAG) and enhanced the content of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) and docosahexaenoic acid (DHA) in M. elongata. To increase TAG productivity in the alga, we developed an effective reduced nitrogen supply regime based on ammonium in environmental photobioreactors (ePBRs). Under optimized conditions, N. oceanica produced high levels of TAG that could be indirectly monitored by following chlorophyll content. Combining N. oceanica and M. elongata to initiate bio-flocculation yielded high levels of TAG and total fatty acids, ~15% and 22% of total dry weight (DW), respectively, as well as high levels of PUFAs. Genetic engineering N. oceanica for higher TAG content in nutrient-replete medium was accomplished by overexpressing DGTT5, a gene encoding the type II acyl-CoA:diacylglycerol acyltransferase 5. Combined with bioflocculation this approach led to increased production of TAG under nutrient replete conditions (~10% of DW) compared to the wild type (~6% of DW). Conclusions: The combined use of M. elongata and N. oceanica with available genomes and genetic engineering tools for both species opens up new avenues to improve bio-fuel productivity and allows the engineering of polyunsaturated fatty acids. Keywords: Microalgae, Filamentous fungi, Flocculation, Cell wall interaction, Biofuel, Nitrogen starvation, Polyunsaturated fatty acid, Triacylglycerolmore » « less
