With its high nutritional value and productivity, Italian ryegrass as a biomass feedstock constantly supplies rumen degradable nitrogen and digestible fiber to ruminants. However, biofuel production is easily reduced during ensiling due to the high-moisture content of Italian ryegrass, leading to economic losses. Lactic acid bacteria inoculants could improve lignocellulosic degradation and fermentation quality and decrease dry matter loss during the bioprocessing of silage. Therefore, this study analyzed the effects of Lactobacillus buchneri TSy1-3 (HE), Lactobacillus rhamnosus BDy3-10 (HO), and the combination of HE and HO (M) on fermentation quality, bacterial community and metabolome in high-moisture Italian ryegrass silage during ensiling. The results showed that the pH value was significantly lower in the HO groups than in the other treatments at the end of ensiling, and the dry matter and acetic acid contents were significantly higher in the HO group than in the other inoculated groups. All inoculants decreased the diversity of the bacterial community and significantly increased the relative abundance of Lactobacillus. Inoculation with HO significantly improved the concentrations of organic acids, dipeptides, ferulic acid, apigenin, and laricitrin. Compared with Lactobacillus buchneri TSy1-3 (HE), HO significantly upregulated the flavonoid compounds in the flavone and flavonol biosynthesis pathway. Overall, these findings suggest that inoculation with HO was beneficial for the development of Italian ryegrass as a biomass feedstock, improving fermentation quality, accelerating changes in bacterial community composition and increasing biofunctional metabolites in high-moisture Italian ryegrass silage.

Increasing seed oil content is the most important breeding goal in Brassica napus, and phenotyping is crucial to dissect its genetic basis in crops. To date, QTL mapping for oil content has been based on whole seeds, and the lipid distribution is far from uniform in different tissues of seeds in B. napus. In this case, the phenotype based on whole seeds was unable to sufficiently reveal the complex genetic characteristics of seed oil content. Here, the three-dimensional (3D) distribution of lipid was determined for B. napus seeds by magnetic resonance imaging (MRI) and 3D quantitative analysis, and ten novel oil content-related traits were obtained by subdividing the seeds. Based on a high-density genetic linkage map, 35 QTLs were identified for 4 tissues, the outer cotyledon (OC), inner cotyledon (IC), radicle (R) and seed coat (SC), which explained up to 13.76% of the phenotypic variation. Notably, 14 tissue-specific QTLs were reported for the first time, 7 of which were novel. Moreover, haplotype analysis showed that the favorable alleles for different seed tissues exhibited cumulative effects on oil content. Furthermore, tissue-specific transcriptomes revealed that more active energy and pyruvate metabolism influenced carbon flow in the IC, OC and R than in the SC at the early and middle seed development stages, thus affecting the distribution difference in oil content. Combining tissue-specific QTL mapping and transcriptomics, 86 important candidate genes associated with lipid metabolism were identified that underlie 19 unique QTLs, including the fatty acid synthesis rate-limiting enzyme-related gene CAC2, in the QTLs for OC and IC. The present study provides further insight into the genetic basis of seed oil content at the tissue-specific level. Three-dimensional phenotyping, QTL identification with a high-density genetic linkage map and transcriptomic analysis were combined to dissect the genetic architecture of oil content in different seed tissues of Brassica napus.

Rapid drying, cost-effective and safe, will increase the viability of using microalgae for several bio-industrial applications. In this study, five different drying techniques of microalgal biomass were investigated. These include freeze drying, oven drying, air drying, sun drying, and microwave drying. Morphology, metabolite content, FAME profiling, chlorophyll content, total organic carbon, and total nitrogen were analyzed. Results showed that the freeze-drying technique preserves the highest amounts of chlorophyll, proteins, and lipids. Oven drying underperformed as it retained the lowest amount of chlorophyll, protein, and lipid content. More importantly, FAME profiling results showed that air drying was the best technique in maintaining the highest amount of polyunsaturated fatty acids and more specifically docosahexaenoic acid (DHA). Furthermore, this process requires the least capital and energy needs. The findings from this study confirmed that the drying technique affects the microalga biomass quality.

Rapid and effective consumption of d-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous d-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming d-xylose, growth inhibition occurs at higher d-xylose concentrations, even abolishing growth completely at 8% d-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in d-xylose metabolism is the phosphorylation of d-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high d-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high d-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust d-xylose metabolism.

With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.

Light is a key regulatory factor for photosynthesis and metabolism of microalgae. The diatom Phaeodactylum tricornutum is capable of exhibiting metabolic flexibility in response to light fluctuations. However, the metabolic switching and underlying molecular mechanisms upon illumination transitions remain poorly understood for this industrially relevant marine alga. To address these, the physiochemical and molecular responses of P. tricornutum upon high light (HL) and recovery (HLR) were probed. Upon HL, P. tricornutum exhibited quick responses, including decreases in cell division, major light harvesting pigments (e.g., chlorophyll a, β-carotene, and fucoxanthin), chloroplastidic membrane lipids (e.g., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol), and long-chain polyunsaturated fatty acids (e.g., C20:5), as well as increases in carbohydrates and neutral lipids particularly triacylglycerol. During HLR stage when the stress was removed, these physiochemical phenotypes were generally recovered, indicative of a rapid and reversible changes of P. tricornutum to cope with illumination transitions for survival and growth. Through the integrated analysis with time-resolved transcriptomics, we revealed the transcriptional control of photosynthesis and carbon metabolism in P. tricornutum responding to HL, which could be reversed more or less during the HLR stage. Furthermore, we highlighted key enzymes involved in carotenoid biosynthesis and lipid metabolism of P. tricornutum and identified monooxygenases putatively responsible for catalyzing the ketolation step towards fucoxanthin synthesis from neoxanthin. The detailed profiling of physiochemical and transcriptional responses of P. tricornutum to HL-HLR treatments advances our understanding on the adaption of the alga to illumination transitions and provides new insights into engineering of the alga for improved production of value-added carotenoids and lipids.

l-Methionine is the only bulk amino acid that has not been industrially produced by the fermentation method. Due to highly complex and strictly regulated biosynthesis, the development of microbial strains for high-level l-methionine production has remained challenging in recent years. By strengthening the l-methionine terminal synthetic module via site-directed mutation of l-homoserine O-succinyltransferase (MetA) and overexpression of metAfbr, metC, and yjeH, l-methionine production was increased to 1.93 g/L in shake flask fermentation. Deletion of the pykA and pykF genes further improved l-methionine production to 2.51 g/L in shake flask fermentation. Computer simulation and auxotrophic experiments verified that during the synthesis of l-methionine, equimolar amounts of l-isoleucine were accumulated via the elimination reaction of cystathionine γ-synthetase MetB due to the insufficient supply of l-cysteine. To increase the supply of l-cysteine, the l-cysteine synthetic module was strengthened by overexpression of cysEfbr, serAfbr, and cysDN, which further increased the production of l-methionine by 52.9% and significantly reduced the accumulation of the byproduct l-isoleucine by 29.1%. After optimizing the addition of ammonium thiosulfate, the final metabolically engineered strain MET17 produced 21.28 g/L l-methionine in 64 h with glucose as the carbon source in a 5 L fermenter, representing the highest l-methionine titer reported to date. In this study, a high-efficiency strain for l-methionine production was derived from wild-type Escherichia coli W3110 by rational metabolic engineering strategies, providing an efficient platform for the industrial production of l-methionine.

Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and β-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of β-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.

To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation. Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Lignocellulose-derived aldehyde inhibitors seriously blocked the biorefinery of biofuels and biochemicals. To date, the economic production of lignocellulose-based products heavily relied on high productivities of fermenting strains. However, it was expensive and time-consuming for the achievable rational modification to strengthen stress tolerance robustness of aldehyde inhibitors. Here, it aimed to improve aldehyde inhibitors tolerance and cellulosic bioethanol fermentability for the chassis Zymomonas mobilis ZM4 pretreated using energy-efficient and eco-friendly cold plasma. It was found that bioethanol fermentability was weaker in CSH (corn stover hydrolysates) than that in synthetic medium for Z. mobilis, and thus was attributed to the inhibition of the lignocellulose-derived aldehyde inhibitors in CSH. Convincingly, it further confirmed that the mixed aldehydes severely decreased bioethanol accumulation through additional aldehydes supplementary assays in synthetic medium. After assayed under different processing time (10–30 s), discharge power (80–160 W), and working pressure (120–180 Pa) using cold atmosphere plasma (CAP), it achieved the increased bioethanol fermentability for Z. mobilis after pretreated at the optimized parameters (20 s, 140 W and 165 Pa). It showed that cold plasma brought about three mutation sites including ZMO0694 (E220V), ZMO0843 (L471L) and ZMO0843 (P505H) via Genome resequencing-based SNPs (single nucleotide polymorphisms). A serial of differentially expressed genes (DEGs) were further identified as the potential contributors for stress tolerance via RNA-Seq sequencing, including ZMO0253 and ZMO_RS09265 (type I secretion outer membrane protein), ZMO1941 (Type IV secretory pathway protease TraF-like protein), ZMOr003 and ZMOr006 (16S ribosomal RNA), ZMO0375 and ZMO0374 (levansucrase) and ZMO1705 (thioredoxins). It enriched cellular process, followed by metabolic process and single-organism process for biological process. For KEGG analysis, the mutant was also referred to starch and sucrose metabolism, galactose metabolism and two-component system. Finally, but interestingly, it simultaneously achieved the enhanced stress tolerance capacity of aldehyde inhibitors and bioethanol fermentability in CSH for the mutant Z. mobilis. Of several candidate genetic changes, the mutant Z. mobilis treated with cold plasma was conferred upon the facilitated aldehyde inhibitors tolerance and bioethanol production. This work would provide a strain biocatalyst for the efficient production of lignocellulosic biofuels and biochemicals.

The development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C–C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work. A one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 ℃, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction. With EOAB and K2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE.

Plant hemoglobin shows great potential as a food additive to circumvent the controversy of using animal materials. Microbial fermentation with engineered microorganisms is considered as a promising strategy for sustainable production of hemoglobin. As an endotoxin-free and GRAS (generally regarded as safe) bacterium, Corynebacterium glutamicum is an attractive host for hemoglobin biosynthesis. Herein, C. glutamicum was engineered to efficiently produce plant hemoglobin. Hemoglobin genes from different sources including soybean and maize were selected and subjected to codon optimization. Interestingly, some candidates optimized for the codon usage bias of Escherichia coli outperformed those for C. glutamicum regarding the heterologous expression in C. glutamicum. Then, saturated synonymous mutation of the N-terminal coding sequences of hemoglobin genes and fluorescence-based high-throughput screening produced variants with 1.66- to 3.45-fold increase in hemoglobin expression level. To avoid the use of toxic inducers, such as isopropyl-β-d-thiogalactopyranoside, two native inducible expression systems based on food additives propionate and gluconate were developed. Promoter engineering improved the hemoglobin expression level by 2.2- to 12.2-fold. Combination of these strategies and plasmid copy number modification allowed intracellular production of hemoglobin up to approximately 20% of total protein. Transcriptome and proteome analyses of the hemoglobin-producing strain revealed the cellular response to excess hemoglobin accumulation. Several genes were identified as potential targets for further enhancing hemoglobin production. In this study, production of plant hemoglobin in C. glutamicum was systematically engineered by combining codon optimization, promoter engineering, plasmid copy number modification, and multi-omics-guided novel target discovery. This study offers useful design principles to genetically engineer C. glutamicum for the production of hemoglobin and other recombinant proteins.

The marine alga Nannochloropsis oceanica, an emerging model belonging to Heterokont, is considered as a promising light-driven eukaryotic chassis for transforming carbon dioxide to various compounds including carotenoids. Nevertheless, the carotenogenic genes and their roles in the alga remain less understood and to be further explored. Here, two phylogenetically distant zeaxanthin epoxidase (ZEP) genes from N. oceanica (NoZEP1 and NoZEP2) were functionally characterized. Subcellular localization experiment demonstrated that both NoZEP1 and NoZEP2 reside in the chloroplast yet with differential distribution patterns. Overexpression of NoZEP1 or NoZEP2 led to increases of violaxanthin and its downstream carotenoids at the expense of zeaxanthin in N. oceanica, with the extent of changes mediated by NoZEP1 overexpression being greater as compared to NoZEP2 overexpression. Suppression of NoZEP1 or NoZEP2, on the other hand, caused decreases of violaxanthin and its downstream carotenoids as well as increases of zeaxanthin; similarly, the extent of changes mediated by NoZEP1 suppression was larger than that by NoZEP2 suppression. Interestingly, chlorophyll a dropped following violaxanthin decrease in a well-correlated manner in response to NoZEP suppression. The thylakoid membrane lipids including monogalactosyldiacylglycerol also correlated with the violaxanthin decreases. Accordingly, NoZEP1 suppression resulted in more attenuated algal growth than NoZEP2 suppression did under either normal light or high light stage. The results together support that both NoZEP1 and NoZEP2, localized in the chloroplast, have overlapping roles in epoxidating zeaxanthin to violaxanthin for the light-dependent growth, yet with NoZEP1 being more functional than NoZEP2 in N. oceanica. Our study provides implications into the understanding of carotenoid biosynthesis and future manipulation of N. oceanica for carotenoid production.

Sugarcane trash (SCT) represents up to 18% of the aboveground biomass of sugarcane, surpassing 28 million tons globally per year. The majority of SCT is burning in the fields. Hence, efficient use of SCT is necessary to reduce carbon dioxide emissions and global warming and establish agro-industrial biorefineries. Apart from its low costs, conversion of whole biomass with high production efficiency and titer yield is mandatory for effective biorefinery systems. Therefore, in this study, we developed a simple, integrated method involving a single step of glycerolysis pretreatment to produce antiviral glycerolysis lignin (AGL). Subsequently, we co-fermented glycerol with hydrolyzed glucose and xylose to yield high titers of bioethanol. SCT was subjected to pretreatment with microwave acidic glycerolysis with 50% aqueous (aq.) glycerol (MAG50); this pretreatment was optimized across different temperature ranges, acid concentrations, and reaction times. The optimized MAG50 (opMAG50) of SCT at 1:15 (w/v) in 1% H2SO4, 360 µM AlK(SO4)2 at 140 °C for 30 min (opMAG50) recovered the highest amount of total sugars and the lowest amount of furfural byproducts. Following opMAG50, the soluble fraction, i.e., glycerol xylose-rich solution (GXRS), was separated by filtration. A residual pulp was then washed with acetone, recovering 7.9% of the dry weight (27% of lignin) as an AGL. AGL strongly inhibited the replication of encephalomyocarditis virus (EMCV) in L929 cells without cytotoxicity. The pulp was then saccharified in yeast peptone medium by cellulase to produce a glucose concentration similar to the theoretical yield. The total xylose and arabinose recoveries were 69% and 93%, respectively. GXRS and saccharified sugars were combined and co-fermented through mixed cultures of two metabolically engineered Saccharomyces cerevisiae strains: glycerol-fermenting yeast (SK-FGG4) and xylose-fermenting yeast (SK-N2). By co-fermenting glycerol and xylose with glucose, the ethanol titer yield increased to 78.7 g/L (10% v/v ethanol), with a 96% conversion efficiency. The integration of AGL production with the co-fermentation of glycerol, hydrolyzed glucose, and xylose to produce a high titer of bioethanol paves an avenue for the use of surplus glycerol from the biodiesel industry for the efficient utilization of SCT and other lignocellulosic biomasses.

At present, the conventional methods for determining photosynthetic products of microalgae are usually based on a large number of cell mass to reach the measurement baseline, and the result can only reveal the average state at the population level, which is not feasible for large-scale and rapid screening of specific phenotypes from a large number of potential microalgae mutants. In recent years, single-cell Raman spectra (SCRS) has been proved to be able to rapidly and simultaneously quantify the biochemical components of microalgae. However, this method has not been reported to analyze the biochemical components of Cyclotella cryptica (C. cryptica). Thus, SCRS was first attempt to determine these four biochemical components in this diatom. The method based on SCRS was established to simultaneously quantify the contents of polysaccharide, total lipids, protein and Chl-a in C. cryptica, with thirteen Raman bands were found to be the main marker bands for the diatom components. Moreover, Partial Least Square Regression (PLSR) models based on full spectrum can reliably predict these four cellular components, with Pearson correlation coefficient for these components reached 0.949, 0.904, 0.801 and 0.917, respectively. Finally, based on SCRS data of one isogenic sample, the pairwise correlation and dynamic transformation process of these components can be analyzed by Intra-ramanome Correlation Analysis (IRCA), and the results showed silicon starvation could promote the carbon in C. cryptica cells to flow from protein and pigment metabolism to polysaccharide and lipid metabolism. First, method for the simultaneous quantification of the polysaccharide, total lipid, protein and pigment in single C. cryptica cell are established. Second, the instant interconversion of intracellular components was constructed through IRCA, which is based on data set of one isogenic population and more precision and timeliness. Finally, total results indicated that silicon deficiency could promote the carbon in C. cryptica cells to flow from protein and pigment metabolism to polysaccharide and lipid metabolism.

Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures. In slow-growing anaerobic chemostat cultures (D = 0.05 h−1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h−1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h−1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h−1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower. Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Esters are widely used in food, energy, spices, chemical industry, etc., becoming an indispensable part of life. However, their production heavily relies on the fossil energy industry, which presents significant challenges associated with energy shortages and environmental pollution. Consequently, there is an urgent need to identify alternative green methods for ester production. One promising solution is biosynthesis, which offers sustainable and environmentally friendly processes. In ester biosynthesis, alcohol acyltransferases (AATs) catalyze the condensation of acyl-CoAs and alcohols to form esters, enabling the biosynthesis of nearly 100 different kinds of esters, such as ethyl acetate, hexyl acetate, ethyl crotonate, isoamyl acetate, and butyl butyrate. However, low catalytic efficiency and low selectivity of AATs represent the major bottlenecks for the biosynthesis of certain specific esters, which should be addressed with protein molecular engineering approaches before practical biotechnological applications. This review provides an overview of AAT enzymes, including their sequences, structures, active sites, catalytic mechanisms, and metabolic engineering applications. Furthermore, considering the critical role of AATs in determining the final ester products, the current research progresses of AAT modification using protein molecular engineering are also discussed. This review summarized the major challenges and prospects of AAT enzymes in ester biosynthesis.

After centuries of heavy reliance on fossil fuel energy, the world suffers from an energy crisis and global warming, calling for carbon emission reduction and a transition to clean energy. Microalgae have attracted much attention as a potential feedstock for biofuel production due to their high triacylglycerol content and CO2 sequestration ability. Many diacylglycerol acyltransferases (DGAT) species have been characterized, which catalyze the final committed step in triacylglycerol biosynthesis. However, the detailed structure–function features of DGATs and the role of the interactions among DGAT proteins in lipid metabolism remained largely unknown. In this study, the three characterized DGATs of Auxenochlorella protothecoides 2341 showed distinct structural and functional conservation. Functional complementation analyses showed that ApDGAT1 had higher activity than ApDGAT2b in yeast and model microalgae, and ApDGAT2a had no activity in yeast. The N-terminus was not essential to the catalysis function of ApDGAT1 but was crucial to ApDGAT2b as its enzyme activity was sensitive to any N-terminus modifications. Similarly, when acyl-CoA binding proteins (ACBPs) were fused to the N-terminus of ApDGAT1 and ApDGAT2b, zero and significant activity changes were observed, respectively. Interestingly, the ApACBP3 + ApDGAT1 variant contributed to higher oil accumulation than the original DGAT1, and ApACBP1 + ApDGAT1 fusion boosted oleic acid content in yeast. Overexpression of the three DGATs and the variation of ApACBP3 + ApDGAT1 increased the content of C18:1 of Chlamydomonas reinhardtii CC-5235. Significantly, ApDGAT1 interacted with itself, ApDGAT2b, and ApACBP1, which indicated that these three lipid metabolic proteins might have been a part of a dynamic protein interactome that facilitated the enrichment of oleic acid. This study provided new insights into the functional and structural characteristics of DGATs and elucidated the importance of these physical interactions in potential lipid channeling.

Rice (Oryza sativa) straw is a common waste product that represents a considerable amount of bound energy. This energy can be used for biogas production, but the rate and level of methane produced from rice straw is still low. To investigate the potential for an increased biogas production from rice straw, we have here utilized WRINKLED1 (WRI1), a plant AP2/ERF transcription factor, to increase triacylglycerol (TAG) biosynthesis in rice plants. Two forms of Arabidopsis thaliana WRI1 were evaluated by transient expression and stable transformation of rice plants, and transgenic plants were analyzed both for TAG levels and biogas production from straw. Both full-length AtWRI1, and a truncated form lacking the initial 141 amino acids (including the N-terminal AP2 domain), increased fatty acid and TAG levels in vegetative and reproductive tissues of Indica rice. The stimulatory effect of the truncated AtWRI1 was significantly lower than that of the full-length protein, suggesting a role for the deleted AP2 domain in WRI1 activity. Full-length AtWRI1 increased TAG levels also in Japonica rice, indicating a conserved effect of WRI1 in rice lipid biosynthesis. The bio-methane production from rice straw was 20% higher in transformants than in the wild type. Moreover, a higher producing rate and final yield of methane was obtained for rice straw compared with rice husks, suggesting positive links between methane production and a high amount of fatty acids. Our results suggest that heterologous WRI1 expression in transgenic plants can be used to improve the metabolic potential for bioenergy purposes, in particular methane production.

Many fresh water bodies face a great challenge of an invasive weed called water hyacinth (WH) which has great impacts on the environment, ecology, and society. Food and Agriculture Organization (FAO) estimates that over nine million tons of Fish wastes (FW) are thrown away each year. The fish waste generated poses environmental and health hazards because in most cases it is either disposed into pits or discarded onto the open grounds. Both WH and FW are potential substrates for biogas production. However, utilization of FW substrate alone has a limitation of producing a lot of amounts of volatile fatty acids (VFAs) and ammonia. Their accumulation in the digester inhibits substrate digestion. Consequently, as stand-alone it is not suitable for anaerobic digestion (AD). This can be overcome by co-digestion with a substrate like WH which has high carbon to nitrogen (C/N) ratio prior to biodigestion. Experimental variable levels for biogas were substrate ratio (WH:FW, 25–75 g), inoculum concentration (IC, 5–15 g/250 mL), and dilution (85–95 mL). Design-Expert 13 was used for optimization and results analysis. Response surface methodology (RSM) was used to examine the effects of operating parameters and identify optimum values for biogas yield. Optimum values for maximum biogas with the highest methane yield of 68% were found to be WH:FW ratio, 25:75 g, 15 g of IC, and 95 mL for dilution. The yield was 16% and 32% greater than FW and WH mono-digestion, respectively. The biogas yield was expressed as a function of operating variables using a quadratic equation. The model was significant (P  中文翻译： 鱼类废物和水葫芦厌氧共消化沼气生产的优化 许多淡水体面临着一种名为水葫芦（WH）的入侵杂草的巨大挑战，它对环境、生态和社会产生巨大影响。联合国粮食及农业组织 (FAO) 估计，每年有超过 900 万吨鱼类废物 (FW) 被丢弃。产生的鱼类废物会对环境和健康造成危害，因为在大多数情况下，它要么被丢弃到坑中，要么被丢弃在露天场所。WH 和 FW 都是沼气生产的潜在底物。然而，单独利用 FW 底物存在产生大量挥发性脂肪酸 (VFA) 和氨的局限性。它们在消化器中的积累会抑制底物消化。因此，单独使用时它不适合厌氧消化 (AD)。这可以通过在生物消化之前与具有高碳氮 (C/N) 比的 WH 等底物共消化来克服。沼气的实验变量水平是底物比率（WH:FW，25-75 g）、接种物浓度（IC，5-15 g/250 mL）和稀释度（85-95 mL）。Design-Expert 13 用于优化和结果分析。响应面法（RSM）用于检查操作参数的影响并确定沼气产量的最佳值。发现最大沼气和最高甲烷产率 68% 的最佳值为 WH:FW 比例、25:75 g、IC 15 g 和稀释 95 mL。产量分别比 FW 和 WH 单一消化高 16% 和 32%。使用二次方程将沼气产量表示为操作变量的函数。该模型显着（P < 0.05）。所有因素对沼气均具有显着的线性和二次效应，只有两个因素的交互作用才显着。99.9% 的决定系数 (R2) 证实了模型与实验变量的良好拟合。