C/N Ratio and Specific Growth Rate Plays Important Role on Enhancing Isoprene Production in Recombinant Escherichia coli.

Effect of fermentation parameters such as C/N ratio, specific growth rate, phosphate limitation, and plasmid instability on enhancing isoprene production is the focus of the current study. Isoprene productivity in the recombinant Escherichia coli K12_MVA strain showed a bell-shaped relationship with specific growth rate in bioreactor studies with isoprene volumetric productivity peaking at 0.35/h. This behavior was depicted by a production inhibition kinetic model which envisaged a serious competition between the cellular growth, acetic acid production, and isoprene biosynthesis. The model equation derived showed a reasonable fit with the experimental values. Judicious control of the growth rates and acetate accumulation by optimizing C/N ratio, phosphate concentration, and intermittent feeding strategy resulted in maximizing the carbon flux towards isoprene. Plasmid instability caused by metabolic burden posed by the presence of dual plasmids on the bacteria was simulated using first-order degradation kinetics. The experimental plasmid loss trend was in accordance with the model simulated trend, where higher plasmid loss correlated with higher specific growth rates. Modulating the growth rate, acetate accumulation, and plasmid instability resulted in achieving maximum isoprene volumetric productivity of 1.125 g/l/h with 46.67% of carbon flux towards isoprene and a isoprene titre of 18 g/l in 16 h fermentation run.

Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Enhanced Cadaverine Production by Engineered Escherichia coli Using Soybean Residue Hydrolysate (SRH) as a Sole Nitrogen Source.

An economical source of nitrogen is one of the major limiting factors for sustainable cadaverine production. The utilization potential of soybean residue for enhanced cadaverine production by engineered Escherichia coli DFC1001 was investigated in this study. The SRH from soybean residue could get the protein extraction rate (PE) of 67.51% and the degree of protein hydrolysis (DH) of 22.49%. The protein molecular weights in SRH were mainly distributed in 565 Da (72.28%) and 1252 Da (17.11%). These proteins with small molecular weights and concentrated molecular weight distribution were favorable to be transformed by engineered E. coli DFC1001, and then SRH replaced completely yeast powder as an only nitrogen source for cadaverine production. The maximum cadaverine productivity was 0.52 g/L/h, achieved with a constant speed feeding strategy in the optimized SRH fermentation medium containing an initial total sugar concentration of 30 g/L and exogenous added minerals, which indicated that soybean residue could be a potential feedstock for economic cadaverine production.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage compounds. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most valuable biopolymers because of its superior mechanical properties. Here, we developed a bioprocess utilizing recombinant Bacillus megaterium strain for PHBV over-production from glucose, without any precursor addition. PHA production was performed in a controlled bioreactor by batch and fed-batch modes using wild-type B. megaterium and rec-B. megaterium cells overexpressing the native phaC gene. The effect of oxygen transfer rate on biomass formation and PHA accumulation was also investigated, under different dissolved oxygen levels. Structural and thermal properties of PHA were characterized by GC-FID, 1H-NMR, TGA and DSC analyses. Significantly, the copolymer produced from glucose as the carbon source in rec-B. megaterium was composed of 58 mol% of 3-hydroxyvalerate monomers. After 66 h, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ0 = 0.1 h-1 produced the highest CDW (7.7 g L-1) and PHA concentration (6.1 g L-1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.8 g g-1.

Limited oxygen conditions as an approach to scale-up and improve D and L-lactic acid production in mineral media and avocado seed hydrolysates with metabolically engineered Escherichia coli.

The effectiveness of micro-aeration on lactate (LA) production by metabolically engineered Escherichia coli was evaluated in 1 L bioreactors containing mineral media and glucose (70 g/L). Volumetric oxygen transfer coefficients (kLa) between 12.6 and 28.7 h-1 increased the specific growth rate (µ) and volumetric productivity (QLA) by 300 and 400%, respectively, without a significant decrease in lactate yield (YLA), when compared with non-aerated fermentations. A kLa of 12.6 h-1 was successfully used as a criterion to scale-up the production of L and D-lactate from 1 to 11 and 130 L. Approximately constant QLA and YLA values were obtained throughout the fermentation scale-up process. Furthermore, a D-lactogenic fermentation was carried out in 1 L bioreactors using avocado seed hydrolysate as a culture medium under the same kLa value, displaying high QLA and YLA.

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed "starch-mannose-fermentation" biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Controlling the Implementation of Transgenic Microbes: Are We Ready for What Synthetic Biology Has to Offer?

Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority. Here, we present an in-depth summary of biological containment systems for micro-organisms published to date, including the production of a genetic firewall through genome recoding and physical containment of microbes using auxotrophies, regulation of essential genes, and expression of toxic genes. The level of containment required to consider a transgenic organism suitable for deployment is discussed, as well as standards of practice for developing new containment systems.

Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803.

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely 'UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that 'UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO2 into excreted FAME.

An auto-inducible expression and high cell density fermentation of Beefy Meaty Peptide with Bacillus subtilis.

Currently, some cases about the expression of flavor peptides with microorganisms were reported owing to the obvious advantages of biological expression over traditional methods. However, beefy meaty peptide (BMP), the focus of umami peptides, has neither been concerned in its safe expression nor its overproduction in fermenter. In this study, multi-copy BMP (8BMP) was successfully auto-inducibly expressed and efficiently produced in Bacillus subtilis 168. First, 8BMP was successfully auto-inducibly expressed with srfA promoter in B. subtilis 168. Further, the efficient production of 8BMP was researched in a 5-L fermenter: the fermentation optimized by Pontryagin's maximum principle obtained the highest 8BMP yield (3.16 g/L), which was 1.2 times and 1.8 times than that of two-stage feeding cultivation (2.67 g/L) and constant-rate feeding cultivation (1.75 g/L), respectively. Overall, the auto-inducible expression of 8BMP in B. subtilis and fermentation with Pontryagin's maximum principle are conductive for overproduction of BMP and other peptides.

Engineering Yarrowia lipolytica for the utilization of acid whey.

Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted ß-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology.

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae.

S-Adenosyl-l-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l-methionine is generally used, the low-cost dl-methionine is more suitable as the substrate for industrial production of SAM. However, d-methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl-methionine, intracellular conversion of d-methionine to l-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d-amino acid-N-acetyltransferase, d-methionine was accumulated in vivo and no N-acetyl-d-methionine production was observed. Further, codon-optimized d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l-phenylalanine dehydrogenase gene (l-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d-methionine to l-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d-methionine. Using the recently developed CRISPR tools, the DAAO and l-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl-methionine.

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 µg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

Tailoring of microbes for the production of high value plant-derived compounds: From pathway engineering to fermentative production.

Plant natural products have been an attracting platform for the isolation of various active drugs and other bioactives. However large-scale extraction of these compounds is affected by the difficulty in mass cultivation of these plants and absence of strategies for successful extraction. Even though, synthesis by chemical method is an alternative method; it is less efficient as their chemical structure is highly complex which involve enantio-selectivity. Thus an alternate bio-system for heterologous production of plant natural products using microbes has emerged. Advent of various omics, synthetic and metabolic engineering strategies revolutionised the field of heterologous plant metabolite production. In this context, various engineering methods taken to synthesise plant natural products are described with an additional focus to fermentation strategies.

The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology.

Harnessing the process of natural selection to obtain and understand new microbial phenotypes has become increasingly possible due to advances in culturing techniques, DNA sequencing, bioinformatics, and genetic engineering. Accordingly, Adaptive Laboratory Evolution (ALE) experiments represent a powerful approach both to investigate the evolutionary forces influencing strain phenotypes, performance, and stability, and to acquire production strains that contain beneficial mutations. In this review, we summarize and categorize the applications of ALE to various aspects of microbial physiology pertinent to industrial bioproduction by collecting case studies that highlight the multitude of ways in which evolution can facilitate the strain construction process. Further, we discuss principles that inform experimental design, complementary approaches such as computational modeling that help maximize utility, and the future of ALE as an efficient strain design and build tool driven by growing adoption and improvements in automation.

Exploiting Hydrogenophaga pseudoflava for aerobic syngas-based production of chemicals.

Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative ß-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H. pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as PgapA2 which proved to yield high expression under heterotrophic and autotrophic conditions. H. pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H. pseudoflava exhibited a growth rate of 0.06 h-1 and biomass specific uptakes rates of 14.2 ±â€¯0.3 mmol H2 gCDW-1 h-1, 73.9 ±â€¯1.8 mmol CO gCDW-1 h-1, and 31.4 ±â€¯0.3 mmol O2 gCDW-1 h-1. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C15 sesquiterpene (E)-α-bisabolene from the C1 carbon source syngas by heterologous expression of the (E)-α-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 ±â€¯8 µg (E)-α-bisabolene L-1 with a volumetric productivity Qp of 1.2 ±â€¯0.2 µg L-1 h-1 and a biomass-specific productivity qp of 13.1 ±â€¯0.6 µg gCDW-1 h-1. The intrinsic properties and the genetic repertoire of H. pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas.

Large-scale production of ursodeoxycholic acid from chenodeoxycholic acid by engineering 7α- and 7ß-hydroxysteroid dehydrogenase.

7α- and 7ß-hydroxysteroid dehydrogenases (HSDHs) are key biocatalysts for the biotransformation of ursodeoxycholic acid (UDCA) from chenodeoxycholic acid (CDCA). Various researches focused on heterogeneously expressed engineering enzymes to epimerize CDCA for UDCA, however not yet applied to further industrial application. In this work, we present the large-scale production of UDCA from CDCA by 7α- and 7ß-HSDH enzymatic synthesis. Engineering enzymes were both successfully heterologous overexpressed in Escherichia coli BL21, and the effect of the enzymatic synthesis was investigated. The mass analysis (MS), IR spectrum, 1H NMR and 13C NMR were used to characterize the product. 500-L fermentor fermentation strategy producing a stable supply of HSDH and large-scale production process of UDCA in dozens kilogram class enabled industrial application.

Enhancement of bioethanol production from Gracilaria verrucosa by Saccharomyces cerevisiae through the overexpression of SNR84 and PGM2.

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations.

Production of pyranoanthocyanins using Escherichia coli co-cultures.

Hydroxyphenyl-pyranoanthocyanins are one of the pyranoanthocyanins found in red wines and some fruit juices. Since they have a fourth ring (pyran or ring D) which provides higher color intensity and exceptional stability toward pH variations in comparison to their anthocyanin precursors, these molecules are one of the most important candidates as natural colorants especially for low- and medium-acidic food and beverages. However, their isolation and characterization are difficult due to their very low concentration. In this study, we co-cultured recombinant E. coli strains to synthesize pyranoanthocyanins with improved titers and yields. To accomplish this task, firstly we engineered 4-vinylphenol and 4-vinylcatechol producer modules then we co-cultured each one of these strains with cyanidin-3-O-glucoside producer recombinant cells to obtain pyranocyanidin-3-O-glucoside-phenol (cyanidin-3-O-glucoside with vinylphenol adduct) and pyranocyanidin-3-O-glucoside-catechol (cyanidin-3-O-glucoside with vinylcatechol adduct). By optimizing the co-culture conditions, we were able to significantly increase final titers and yields, allowing our co-culture approach to easily outperform production of pyranoanthocyanins from red wine. Finally, we demonstrate that the produced pyranoanthocyanins are far more stable than the starting plant-produced cyanidin 3-O-glucoside.

Trends in the Bioremediation of Pharmaceuticals and Other Organic Contaminants Using Native or Genetically Modified Microbial Strains: A Review.

Nowadays, numerous synthetic and semisynthetic chemicals are extensively produced and consequently used worldwide for many different purposes, such as pharmaceuticals, pesticides, hydrocarbons with aromatic rings (known as polycyclic aromatic hydrocarbons, PAHs), multi-substituted biphenyls with halogens (such as polychlorinated biphenyls, PCBs), and many other toxic and persistent chemical species. The presence of the aforementioned xenobiotic substances not only in various environmental matrices (water, air, and soil), but also in biological tissues (organisms) as well as in several compartments of raw or processed food (of fruit, vegetal, and animal origin), has raised global scientific concerns regarding their potential toxicity towards non target organisms including humans. Additionally, the ability of those persistent organic pollutants to be magnified via food consumption (food chain) has become a crucial threat to human health. Microbial degradation is considered an important route influencing the fate of those toxicants in each matrix. The technique of bioremediation, either with microorganisms (native or genetically modified) which are applied directly (in a reactor or in situ), or with cell extracts or purified enzymes preparations, is reported as a low cost and potential detoxification technology for the removal of toxic chemicals. The sources and toxic impacts of target groups of chemicals are briefly presented in the present study, whereas the bioremediation applications for the removal of pharmaceuticals and other organic contaminants using microbial strains are critically reviewed. All the recently published data concerning the genes encoding the relevant enzymes that catalyze the degradation reactions, the mechanisms of reactions and parameters that influence the bioremediation process are discussed. Finally, research needs and future trends in the direction of decontamination are high-lightened.

Metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin.

The anthraquinones endocrocin and emodin are synthesized by a special class of type I NR-PKSs and a discrete MßL-TE. In this work, we first reconstituted a biosynthetic pathway of endocrocin and emodin in S. cerevisiae by combining enzymes from different sources. We functionally characterized a TE-less NR-PKS (SlACAS) and a MßL-TE (SlTE) from S. lycopersici as well as four orthologous MßL-TEs. SlACAS was coexpressed with different MßL-TEs in S. cerevisiae. SlACAS generated the highest amount of endocrocin when coupled with HyTE, the yield was 115.6% higher than that with the native SlTE. To accumulate more emodin, seven decarboxylases with high homology to HyDC were identified and introduced into the biosynthetic pathway. Among these orthologs, AfDC exhibited the highest catalytic activity and the conversion rate reached 98.6%. A double-point mutant acetyl-CoA carboxylase, ACC1S659A, S1157A, was further introduced to increase the production of malonyl-CoA as a precursor of these anthraquinones. The production of endocrocin (233.6 ±â€¯20.3 mg/L) and emodin (253.2 ±â€¯21.7 mg/L) then dramatically increased. We also optimized the carbon source in the medium and conducted fed-batch fermentation with the engineered strains. The titers of endocrocin and emodin obtained were 661.2 ±â€¯50.5 mg/L and 528.4 ±â€¯62.7 mg/L, respectively, which are higher than previously reported. In this work, by screening a small library of orthologous biosynthetic bricks, an efficient biosynthetic pathway of endocrocin and emodin was first created in S. cerevisiae. This study provides a novel metabolic engineering approach for optimization of the production of desired molecules.