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Engineering topics

Altpeter, Fredy

Publications and source records attributed to Altpeter, Fredy.

At least 19 records

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis

Stomata regulate CO 2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (g sw ). However, the quantitative relationship between reduced SD, g sw , and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO 2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C 3 dicots, C 3 monocots, and C 4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r 2 =0.60-0.98), but this response is damped relative to the change in anatomy.

59 BASIC BIOLOGICAL SCIENCES↗

The extent of multiallelic, co‐editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield

Summary Sugarcane ( Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 ( LG1 ) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9‐mediated co‐mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2 n = 100–120, x = 10–12). Next generation sequencing revealed co‐editing frequencies of 7.4%–100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co‐editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi‐row field plots. The co‐edited LG1 mutations were stably maintained in vegetative progenies and the extent of co‐editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co‐editing of LG1 in highly polyploid sugarcane allows fine‐tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

59 BASIC BIOLOGICAL SCIENCES↗

Data for Sustainable Co-Production of Plant Lipids and Cellulosic Sugars from Transgenic Energycane at an Industrially Relevant Scale: A Proof of Concept for Alternative Feedstocks

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

Biomass Analytics↗

Sustainable co-production of plant lipids and cellulosic sugars from transgenic energycane at an industrially relevant scale: A proof of concept for alternative feedstocks

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Here, pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

59 BASIC BIOLOGICAL SCIENCES↗

Triacylglycerol, total fatty acid, and biomass accumulation of metabolically engineered energycane grown under field conditions confirms its potential as feedstock for drop‐in fuel production

Abstract Metabolic engineering for hyperaccumulation of lipids in vegetative tissues of high biomass crops promises a step change in oil yields for the production of advanced biofuels. Energycane is the ideal feedstock for this approach due to its exceptional biomass production and persistence under marginal conditions. Here, we evaluated metabolically engineered energycane with constitutive expression of the lipogenic factors WRINKLED 1 ( WRI 1), DIACYLGLYCEROL ACYLTRANSFERASE 1 ( DGAT 1), and OLEOSIN 1 ( OLE 1) for the accumulation of triacylglycerol (TAG), total fatty acid (TFA), and biomass under field conditions at the University of Florida‐IFAS experiment station near Citra, Florida. TAG and TFA accumulation were highest in leaves (up to 9.9% and 12.9% of DW, respectively), followed by juice from crushed stems, stems, and roots. TAG and TFA accumulation increased up to harvest time and correlated highest with OLE 1 and DGAT 1 expression. Biomass dry weight, TAG, and TFA content differed greatly depending on DGAT 1 and OLE 1 expression in transgenic lines with similar WRI 1 expression. Biomass did not significantly differ between WT and line L2 with DAGT 1 and OLE 1 expressed at low levels and TAG and TFA accumulating to 12‐ and 1.6‐fold that of WT leaves, respectively. In contrast, line L13, with intron‐mediated enhancement of DGAT 1 expression, displayed a 245‐ to 330‐fold increase in TAG and a 4.75‐ to 6.45‐fold increase in TFA content compared with WT leaves and a biomass reduction of 52%. These results provide the basis for developing novel feedstocks for expanding plant lipid production and point to new prospects for advanced biofuels.

59 BASIC BIOLOGICAL SCIENCES↗

Raw data for publication: Cao et al. 2023. GCB-Bioenergy (accepted for publication).

Raw data for publication: Viet Dang Cao, Baskaran Kannan, Guangbin Luo, Hui Liu, John Shanklin, and Fredy Altpeter. 2023. Triacylglycerol, total fatty acid and biomass accumulation of metabolically engineered energycane grown under field conditions. GCB-Bioenergy (accepted for publication).

Bioenergy, energycane, lipids, biodiesel, biofuel,↗

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues

Abstract Background Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE ( DGAT ) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. Results In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT 1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED 1 ( WRI 1) and the TAG protect factor oleosin ( OLE 1). Including an intron in the codon-optimized TmDGAT 1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI 1 (W), DGAT 1 with intron (Di), and OLE 1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction. Conclusions Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels. Graphical abstract

59 BASIC BIOLOGICAL SCIENCES↗

Data for Intron-Mediated Enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 Expression in Energycane Promotes a Step Change for Lipid Accumulation in Vegetative Tissues

Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE ( DGAT ) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 ( WRI1 ) and the TAG protect factor oleosin ( OLE1 ). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction. Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.

Feedstock Production↗

Lipid–enhanced Oilcane does not impact soil carbon dynamics compared with wild–type Sugarcane

The carbon neutral potential of bioenergy relies in part on the ability of feedstocks to sequester carbon (C) in the soil. Sugarcane is one of the most widely used bioenergy crops, yet there remain unknowns about how it impacts soil C dynamics. In addition, Oilcane, a genetically modified version of Sugarcane has been produced to accumulate more energy-dense oils and less soluble lignin, which enhances conversion efficiency but may also impact soil C cycling. Thus, our objectives were to examine the impact of Sugarcane litter decomposition on soil C formation and losses and determine if the genetic modifications to produce Oilcane alter these dynamics. To do this, we incubated bagasse (processed stem litter) and leaf litter from Sugarcane and Oilcane in microcosms with forest soil for 11 weeks. We used differences in natural abundance δ 13 C between C 3 forest soil and C 4 litter to trace the fate of the litter into respiratory losses as well as stable and unstable soil C pools. Our results show that genetic modifications to Oilcane did not substantially alter soil C dynamics. Sugarcane and Oilcane litter both led to net soil C gains dominated by an accumulation of the added litter as unstable, particulate organic C (POC). Oilcane litter led to small but significantly greater net soil C gains than Sugarcane litter due to greater POC formation, but the formation of stable, mineral associated organic matter (MAOC) did not differ between crop types. Sugarcane and Oilcane had opposing effects on tissue type where Sugarcane bagasse formed more MAOC, while Oilcane leaves preferentially remained as POC which may have important management implications. These results suggest that genetic modifications to Sugarcane will not significantly impact soil C dynamics; however, this may not be universal to other crops particularly if modifications lead to greater differences in litter chemistry.

09 BIOMASS FUELS↗

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production

Abstract Oilcane is a metabolically engineered sugarcane ( Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI 1 transgene. The WRI 1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.

09 BIOMASS FUELS↗

Targeted mutagenesis with sequence–specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome–editing platforms employ sequence–specific nucleases (SSNs) to generate DNA double–stranded breaks at their target gene. Such DNA breaks are typically repaired via the error–prone nonhomologous end–joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end–use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN–mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

60 APPLIED LIFE SCIENCES↗

Bioprocessing, Recovery, and Mass Balance of Vegetative Lipids from Metabolically Engineered “Oilcane” Demonstrates Its Potential as an Alternative Feedstock for Drop-In Fuel Production

Plant oils are increasingly in demand as renewable feedstocks for biodiesel and biochemicals. Currently, oilseeds are the primary source of plant oils. Although the vegetative tissues of plants express lipid metabolism pathways, they do not hyper- accumulate lipids. Elevated synthesis, storage, and accumulation of lipids in vegetative tissues have been achieved by metabolic engineering of sugarcane to produce “oilcane”. This study evaluates the potential of oilcane as a renewable feedstock for the co- production of lipids and fermentable sugars. Oilcane was grown under favorable climatic and field conditions in Florida (FLOC) as well as during an abbreviated growing season, outside its typical growing region, in Illinois (ILOC). The potential lipid yield of 0.35 tons/ha was projected from the hyperaccumulation of fatty acids in the stored vegetative biomass of FLOC, which is approaching the lipid yield of soybean (0.44 tons/ha). Processing of the vegetative tissues of oilcane recovered 0.20 tons/ha, which represents the recovery of 55% of the total lipids from FLOC. Chemical-free hydrothermal bioprocessing of ILOC and FLOC bagasse and leaves at 180 °C for 10 min prevented the degeneration of in situ plant lipids. Here, this allowed the recovery of lipids at the end of the bioprocess with a major fraction of lipids remaining in the biomass residues after pretreatment and saccharification. Improvements through refined biomass processing, crop management, and metabolic engineering are expected to boost lipid yields and make oilcane a prime feedstock for the production of biodiesel.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Data for Bioprocessing, Recovery, and Mass Balance of Vegetative Lipids from Metabolically Engineered ‘Oilcane’ Demonstrates Its Potential as an Alternative Feedstock for Drop-In Fuel Production

Plant oils are increasingly in demand as renewable feedstocks for biodiesel and biochemicals. Currently, oilseeds are the primary source of plant oils. Although the vegetative tissues of plants express lipid metabolism pathways, they do not hyper-accumulate lipids. Elevated synthesis, storage, and accumulation of lipids in vegetative tissues have been achieved by metabolic engineering of sugarcane to produce “oilcane.” This study evaluates the potential of oilcane as a renewable feedstock for the co-production of lipids and fermentable sugars. Oilcane was grown under favorable climatic and field conditions in Florida (FLOC) as well as during an abbreviated growing season, outside its typical growing region, in Illinois (ILOC). The potential lipid yield of 0.35 tons/ha was projected from the hyperaccumulation of fatty acids in the stored vegetative biomass of FLOC, which is approaching the lipid yield of soybean (0.44 tons/ha). Processing of the vegetative tissues of oilcane recovered 0.20 tons/ha, which represents the recovery of 55% of the total lipids from FLOC. Chemical-free hydrothermal bioprocessing of ILOC and FLOC bagasse and leaves at 180 °C for 10 min prevented the degeneration of in situ plant lipids. This allowed the recovery of lipids at the end of the bioprocess with a major fraction of lipids remaining in the biomass residues after pretreatment and saccharification. Improvements through refined biomass processing, crop management, and metabolic engineering are expected to boost lipid yields and make oilcane a prime feedstock for the production of biodiesel.

Conversion↗

Engineering triacylglycerol accumulation in duckweed ( Lemna japonica )

Summary Duckweeds are amongst the fastest growing of higher plants, making them attractive high‐biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol‐inducible cyan fluorescent protein‐ Arabidopsis WRINKLED1 fusion protein ( CFP‐AtWRI1 ), strong constitutive expression of a mouse diacylglycerol:acyl‐CoA acyltransferase2 ( MmDGAT ), and a sesame oleosin variant ( SiOLE(*) ). Individual expression of each gene increased TAG accumulation by 1‐ to 7‐fold relative to controls, while expression of pairs of these genes increased TAG by 7‐ to 45‐fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45‐fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108‐fold to 8.7% of DW after incubation on medium containing 100 μ m estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three‐fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY‐stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven‐fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

Abstract Background The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. Results Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase 1-2 ( DGAT 1-2) and Oleosin 1 ( OLE 1) in combination with RNAi suppression of SUGAR-DEPENDENT 1 ( SDP 1) and Trigalactosyl diacylglycerol 1 ( TGD 1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson’s correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT 1-2, followed by the level of RNAi suppression for SDP 1. Conclusions This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

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