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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Sustainable strategies to achieve industrial ethanol titers from different bioenergy feedstocks: scale-up approach for better ethanol yield

Hydrothermal pretreatment is a promising approach to lignocellulosic biomass processing for enzymatic hydrolysis and high-yield bioethanol fermentation, as it reduces downstream inhibitor content and the amount of toxic byproducts generated. In this paper, the ethanol yield and productivity of an engineered xylose-fermenting strain of Saccharomyces cerevisiae were tested on lignocellulosic hydrolysates produced with varying citrate buffer concentration, solid loading, supplemental nitrogen source, and feedstock of origin, and a semi-integrated bioprocess which integrates enzymatic hydrolysis and bioethanol fermentation was developed. The greatest ethanol yields (g p /g s ) of 0.490 ± 0.008, 0.460 ± 0.001, 0.420 ± 0.002 and 0.410 ± 0.002 were obtained from bioenergy sorghum (BES), Miscanthus × giganteus (MG), energy cane (EC), and oilcane (OC), respectively. In addition, an equivalent of 291 L, 253.54 L, 257.8 L, and 260.3 L of bioethanol were produced per ton of BES, MG, EC, and OC, respectively, by using urea as a nitrogen source in a bioreactor.

09 BIOMASS FUELS↗
DOE OSTI2025

Integration of Metabolic and Bioprocess Engineering for the Production of B-Ketoadipic Acid from Glucose and Xylose by Pseudomonas Putida

..beta..-Ketoadipic acid is a common intermediate in aerobic microbial aromatic catabolism that can be used as a monomer in performance-advantaged biopolymers. Here, we engineered Pseudomonas putida KT2440 to produce ..beta..-ketoadipate from glucose and xylose, the most prevalent carbohydrates derived from lignocellulosic polysaccharides. With the top-performing strain, P. putida GR038, we conducted bioprocess development using glucose and xylose as substrates in a 2 : 1 molar ratio to mimic lignocellulosic hydrolysate. Fed-batch cultivations achieved a titer of 65.8 g L-1 and a rate of 0.69 g L-1 h-1, with a C-mol yield of 0.52. Application of adsorptive in situ product recovery further improved the effective titer to 92.0 g L-1 and the rate to 0.83 g L-1 h-1, while also improving the downstream purity of ..beta..-ketoadipate from 88.3 wt% to 99.0 wt%. These results show promise towards industrial production of ..beta..-ketoadipate from lignocellulosic sugars.

09 BIOMASS FUELS↗
DOE OSTI2020

Effect of hydrothermal synthesis conditions on up-conversion luminescence intensity of β-NaYF{sub 4} : Er{sup 3+}, Yb{sup 3+} submicron particles

The differences in the luminescence intensities of up-conversion β-NaYF{sub 4} : Er{sup 3+}, Yb{sup 3+} particles synthesised by the hydrothermal method under various synthesis conditions are studied. The results of the study lead to the conclusion that in order to achieve the maximum luminescence intensity in such particles, it is necessary to use ammonium fluoride and a medium with pH = 3. In this case, the length of the particles increases, up to the formation of rod-shaped particles. Based on the data on the size of the coherent scattering region and on microstresses, we can assume that the particles are polycrystals. At the same time, limiting the size of the coherent scattering region is possible due to the defective structure. When the nanoparticles are synthesised in a medium with pH = 3, hydrolysed regions containing OH groups are formed on the crystallite surface. The presence of these groups does not affect the intensity of up-conversion luminescence of submicron-size particles. (paper)

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2022

Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., ‘one pot’), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7–11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4–9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33–9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032–0.131 (g/g) and lipid contents were 13.5–53.7% (g/g). Four strains had significantly higher lipid yields and contents. Finally, one of these strains, which had among the highest lipid yield in this study (0.131 ± 0.007 g/g), has not been previously described in the literature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Developmentally-specific physiological and metabolic responses support drought resilience in switchgrass and constrains biofuel yield

Switchgrass (Panicum virgatum) is a promising bioenergy crop due in part to its resilience to drought stress. However, the significance of drought timing remains poorly understood, both from a plant biology perspective and its impact on downstream biofuel production. This study determines the developmental stage-specific physiological and metabolic responses of switchgrass to drought stress and its implications for biofuel production using a custom-built programmable irrigation system. Vegetative, flowering, and senescence-stage drought significantly reduced carbon dioxide assimilation, and stomatal conductance without affecting biomass yield. Metabolic profiling revealed significant accumulation of glucose, fructose, quinic acid, shikimate and GABA during vegetative-stage drought, while flowering and senescence stages exhibited limited metabolic changes. Similarly, specialized metabolites also displayed distinct developmental patterns, with vegetative-stage drought driving the most pronounced metabolic alterations. Thermochemically-treated and hydrolyzed switchgrass biomass from vegetative-stage drought showed elevated lignocellulose-derived compounds and saponins with the latter most positively correlating with fermentation lag times. Conversely, senescence-stage drought enhanced ethanol yields while lowering saponin levels in the hydrolysates. While vegetative-stage drought enhanced physiological resilience, it compromises downstream biofuel production by introducing fermentation inhibitors, particularly saponins.

biofuel↗
DOE OSTI2020

Regeneration of critical-sized defects, in a goat model, using a dextrin-based hydrogel associated with granular synthetic bone substitute

Abstract The development of injectable bone substitutes (IBS) have obtained great importance in the bone regeneration field, as a strategy to reach hardly accessible defects using minimally invasive techniques and able to fit to irregular topographies. In this scenario, the association of injectable hydrogels and bone graft granules is emerging as a well-established trend. Particularly, in situ forming hydrogels have arisen as a new IBS generation. An in situ forming and injectable dextrin-based hydrogel (HG) was developed, aiming to act as a carrier of granular bone substitutes and bioactive agents. In this work, the HG was associated to a granular bone substitute (Bonelike®) and implanted in goat critical-sized calvarial defects (14 mm) for 3, 6 and 12 weeks. The results showed that HG improved the handling properties of the Bonelike® granules and did not affect its osteoconductive features, neither impairing the bone regeneration process. Human multipotent mesenchymal stromal cells from the umbilical cord, extracellular matrix hydrolysates and the pro-angiogenic peptide LLKKK18 were also combined with the IBS. These bioactive agents did not enhance the new bone formation significantly under the conditions tested, according to micro-computed tomography and histological analysis.

Pereira, Isabel↗
DOE OSTI2023

Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Abstract Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Conversion of poplar biomass into high-energy density tricyclic sesquiterpene jet fuel blendstocks

In an effort to ensure future energy security, reduce greenhouse gas emissions and create domestic jobs, the US has invested in technologies to develop sustainable biofuels and bioproducts from renewable carbon sources such as lignocellulosic biomass. Bio-derived jet fuel is of particular interest as aviation is less amenable to electrification compared to other modes of transportation and synthetic biology provides the ability to tailor fuel properties to enhance performance. Specific energy and energy density are important properties in determining the attractiveness of potential bio-derived jet fuels. For example, increased energy content can give the industry options such as longer range, higher load or reduced takeoff weight. Energy-dense sesquiterpenes have been identified as potential next-generation jet fuels that can be renewably produced from lignocellulosic biomass. We developed a biomass deconstruction and conversion process that enabled the production of two tricyclic sesquiterpenes, epi-isozizaene and prespatane, from the woody biomass poplar using the versatile basidiomycete Rhodosporidium toruloides. We demonstrated terpene production at both bench and bioreactor scales, with prespatane titers reaching 1173.6 mg/L when grown in poplar hydrolysate in a 2 L bioreactor. Additionally, we examined the theoretical fuel properties of prespatane and epi-isozizaene in their hydrogenated states as blending options for jet fuel, and compared them to aviation fuel, Jet A. Our findings indicate that prespatane and epi-isozizaene in their hydrogenated states would be attractive blending options in Jet A or other lower density renewable jet fuels as they would improve viscosity and increase their energy density. Saturated epi-isozizaene and saturated prespatane have energy densities that are 16.6 and 18.8% higher than Jet A, respectively. These results highlight the potential of R. toruloides as a production host for the sustainable and scalable production of bio-derived jet fuel blends, and this is the first report of prespatane as an alternative jet fuel.

09 BIOMASS FUELS↗
DOE OSTI2023

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Engineered Pseudomonas putida KT2440 co-utilizes galactose and glucose

Abstract Background Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose. Results In this work, we integrated a De Ley–Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h −1 . Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose. Conclusions We have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.

09 BIOMASS FUELS↗
DOE OSTI2021

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity

Background: Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass. Results: In this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation. Conclusions: Enzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan.

09 BIOMASS FUELS↗
DOE OSTI2021

Overexpression of the rice BAHD acyltransferase AT10 increases xylan-bound p-coumarate and reduces lignin in Sorghum bicolor

Abstract Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum ( Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p -coumarate ( p CA), a higher amount of p -coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of p CA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound p CA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall p CA remains unchanged indicating that most p CA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound p CA and lignification in cell walls.

09 BIOMASS FUELS↗
DOE OSTI2022

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts

Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2024

Feedstock/pretreatment screening for bioconversion of sugar and lignin streams via deacetylated disc-refining

Recent publications have shown the benefits of deacetylation disc-refining (DDR) as a pretreatment process to deconstruct biomass into sugars and lignin residues. Major advantages of DDR pretreatment over steam and dilute acid pretreatment are the removal of acetyl and lignin during deacetylation. DDR does not generate hydroxymethylfurfural (HMF) and furfural which are commonly produced from steam and dilute acid pretreatments. Acetate, lignin, HMF, and furfural are known inhibitors during enzymatic hydrolysis and fermentation. Another advantage of deacetylation is the production of lignin-rich black liquor, which can be upgraded to other bioproducts. Furthermore, due to the lack of sugar degradation during deacetylation, DDR has significantly less sugar loss than other pretreatment methods. Previous studies for DDR have primarily focused on corn stover, but lacked the investigative studies of other feedstocks. This study was designed to screen various DDR process conditions at pilot scale using three different feedstocks, including corn stover, poplar, and switchgrass. The impact of the pretreatment conditions was evaluated by testing hydrolysates for bioconversion to 2,3-butanediol. Pretreatment of biomass by DDR showed high-conversion-yields and 2,3-BDO fermentation production yields. Techno-economic analysis (TEA) of the pretreatment for biomass to sugar was also developed based on NREL’s Aspen Model. This study shows that the cellulose and hemicellulose in poplar was more recalcitrant than herbaceous feedstocks which ultimately drove up the sugar cost. Switchgrass was also more recalcitrant than corn stover but less than poplar.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Data from High Solids Loading Biorefinery for the Production of Cellulosic Sugars from Bioenergy Sorghum

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two-stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

Conversion↗
DOE OSTI2020

Data from Enhanced 2′-Fucosyllactose Production by Engineered Saccharomyces cerevisiae using Xylose as a Co-Substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli , Saccharomyces cerevisiae , Corynebacterium glutamicum , and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from E. coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Conversion↗