Engineering Candida phangngensis-an oleaginous yeast from the Yarrowia clade-for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction.

Candida phangngensis is an ascomycetous yeast and a phylogenetic relative of the industrial workhorse Yarrowia lipolytica. Here, we report that genetic tools already established for use in the latter organism-including promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system for marker recycle-can be transferred to the newer member of the Yarrowia clade with little or no need for modifications. Using these tools, we engineered C. phangngensis for improved cellulosic lipid production by introducing two heterologous yeast genes. First, overexpression of Saccharomyces cerevisiae ADH6 enhanced in situ detoxification of aldehyde fermentation inhibitors that are generated during biomass pretreatment (e.g. furfural). Subsequently, Y. lipolytica DGA1 expression boosted lipid accumulation in C. phangngensis by pulling additional carbon flux into the triacylglycerol synthesis pathway. In acid-pretreated switchgrass hydrolysate cultures, the final engineered strain JQCP04 showed a 58% decrease in lag time and a 32% increase in lipid titer as compared to wild-type PT1-17. Furthermore, we expect that this study will generate new interest in the highly oleaginous yeast C. phangngensis, which is closely related to a safe, industrial species, and is shown here to be quite amenable for genetic manipulation.

A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate.

Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates of Y. lipolytica identified, only a few of the strains have been studied extensively. Furthermore, 12 other yeast species were recently assigned to the Yarrowia clade, and most are not well characterized in terms of cell growth and lipid accumulation, especially in industrially relevant conditions. In the present study, we investigated biomass and lipid production by 57 yeast isolates, representing all 13 species in the Yarrowia clade, on a non-detoxified dilute acid-pretreated switchgrass hydrolysate under highly aerobic conditions. The objective was to compare yeast physiology during growth in an abundant, low-cost biomass feedstock and to expand diversity of genetically tractable, oleaginous yeasts available for lipid research. Screening of 45 Y. lipolytica isolates demonstrated considerable variation within the species in terms of lipid accumulation (min = 0.1 g/L; max = 5.1 g/L; mean = 2.3 g/L); three strains (NRRL YB-420, YB-419, and YB-392) were especially promising for cellulosic biomass conversion with average improvements of 43, 57, and 64%, respectively, in final lipid titer as compared to control strain W29. Subsequently, evaluation of strains from 13 distinct species in the Yarrowia clade identified Candida phangngensis PT1-17 as the top lipid producer with a maximum titer of 9.8 g/L lipid, which was over twofold higher than the second-best species in the clade (Candida hollandica NRRL Y-48254). A small set of the most promising strains from the screenings was further characterized to evaluate inhibitor tolerance, lipid production kinetics, and fatty acid distribution. We expect that the results of this study will pave the way for new biotechnological applications involving previously overlooked and under-characterized strains within the Yarrowia clade.

Whole cell biosynthesis of a functional oligosaccharide, 2'-fucosyllactose, using engineered Escherichia coli.

BACKGROUND: 2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5'-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase. RESULTS: The gene for FucT2 from Helicobacter pylori was introduced to the GDP-l-fucose producing recombinant E. coli BL21 star(DE3) strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3) strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3) strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3) strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose) corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM) analysis. CONCLUSIONS: The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various foods.

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic "death valley" (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions.

Enhanced hexose fermentation by Saccharomyces cerevisiae through integration of stoichiometric modeling and genetic screening.

In order to determine beneficial gene deletions for ethanol production by the yeast Saccharomyces cerevisiae, we performed an in silico gene deletion experiment based on a genome-scale metabolic model. Genes coding for two oxidative phosphorylation reactions (cytochrome c oxidase and ubiquinol cytochrome c reductase) were identified by the model-based simulation as potential deletion targets for enhancing ethanol production and maintaining acceptable overall growth rate in oxygen-limited conditions. Since the two target enzymes are composed of multiple subunits, we conducted a genetic screening study to evaluate the in silico results and compare the effect of deleting various portions of the respiratory enzyme complexes. Over two-thirds of the knockout mutants identified by the in silico study did exhibit experimental behavior in qualitative agreement with model predictions, but the exceptions illustrate the limitation of using a purely stoichiometric model-based approach. Furthermore, there was a substantial quantitative variation in phenotype among the various respiration-deficient mutants that were screened in this study, and three genes encoding respiratory enzyme subunits were identified as the best knockout targets for improving hexose fermentation in microaerobic conditions. Specifically, deletion of either COX9 or QCR9 resulted in higher ethanol production rates than the parental strain by 37% and 27%, respectively, with slight growth disadvantages. Also, deletion of QCR6 led to improved ethanol production rate by 24% with no growth disadvantage. The beneficial effects of these gene deletions were consistently demonstrated in different strain backgrounds and with four common hexoses. The combination of stoichiometric modeling and genetic screening using a systematic knockout collection was useful for narrowing a large set of gene targets and identifying targets of interest.

Marine macroalgae: an untapped resource for producing fuels and chemicals.

As world energy demand continues to rise and fossil fuel resources are depleted, marine macroalgae (i.e., seaweed) is receiving increasing attention as an attractive renewable source for producing fuels and chemicals. Marine plant biomass has many advantages over terrestrial plant biomass as a feedstock. Recent breakthroughs in converting diverse carbohydrates from seaweed biomass into liquid biofuels (e.g., bioethanol) through metabolic engineering have demonstrated potential for seaweed biomass as a promising, although relatively unexplored, source for biofuels. This review focuses on up-to-date progress in fermentation of sugars from seaweed biomass using either natural or engineered microbial cells, and also provides a comprehensive overview of seaweed properties, cultivation and harvesting methods, and major steps in the bioconversion of seaweed biomass to biofuels.

Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast.

The anticipation for substituting conventional fossil fuels with cellulosic biofuels is growing in the face of increasing demand for energy and rising concerns of greenhouse gas emissions. However, commercial production of cellulosic biofuel has been hampered by inefficient fermentation of xylose and the toxicity of acetic acid, which constitute substantial portions of cellulosic biomass. Here we use a redox balancing strategy to enable efficient xylose fermentation and simultaneous in situ detoxification of cellulosic feedstocks. By combining a nicotinamide adenine dinucleotide (NADH)-consuming acetate consumption pathway and an NADH-producing xylose utilization pathway, engineered yeast converts cellulosic sugars and toxic levels of acetate together into ethanol under anaerobic conditions. The results demonstrate a breakthrough in making efficient use of carbon compounds in cellulosic biomass and present an innovative strategy for metabolic engineering whereby an undesirable redox state can be exploited to drive desirable metabolic reactions, even improving productivity and yield.