Fed-batch Fermentation Research Articles

Green utilization of sorghum straw by coupled hydrothermal pretreatment and cellulase hydrolysis for bacillomycin D production under fed-batch mode

The present work explored the coupled hydrothermal pretreatment and cellulase hydrolysis (HPCH) for production of bacillomycin D from sorghum straws by Bacillus subtilis NS-174. The results indicated that HPCH treated sorghum straws had weaker absorption, heavier mass loss and higher crystallinity, and displayed loose and destructive fiber structures. Degradation of hemicellulose and lignin contributhed to yielding glucose and 68.94g/L of glucose was obtained in HPCH treated hydrolysate. High level of glucose in pretreated sorghum straw hydrolysate was helpful for improving bacillomycin D production. When performed by 3rd round (feeding at 144h) fed-batch fermentation, bacillomycin D production and production rate were obtained to be 2351.07mg/L and 113.36mg/L.h in B. subtilis NS-174, respectively.

Utilization of Aspergillus niger for the fermentative production of azaphilone dye in YEPB medium.

The current research focuses on the production and optimization of a natural yellowish-brown Azaphilone dye using Aspergillus niger. A variety of culture media were tested to ascertain the best conditions for dye synthesis. The formation of the yellowish-brown dye was confirmed by a color shift in the reaction mixture, and UV-Vis spectroscopy detected the dye at 450nm. Static conditions were found to be more favorable than shaking for higher dye yields, and fed-batch fermentation was more effective than batch fermentation. Maximum dye production was achieved after 28days of incubation. Factors such as temperature, pH, and inoculum percentage were shown to influence dye synthesis, with the highest production (2.5ml) occurring at 30°C, pH 7, and a 3% spore suspension in yeast extract peptone broth (YEPB) medium under static conditions. Gas chromatography-mass spectrometry (GC-MS) analysis validated the presence of Azaphilone dye in the culture filtrate. The dye was successfully applied to a pretreated cotton cloth. These findings advance our understanding of optimizing fungal dye production for sustainable and eco-friendly textile coloration applications. This study appears to be the first of its kind to report azaphilone dye production by A. niger in the YEPB medium.

Metabolic engineering of Corynebacterium glutamicum for the production of pyrone and pyridine dicarboxylic acids

Environmental concerns from plastic waste are driving interest in alternative monomers from bio-based sources. Pseudoaromatic dicarboxylic acids are promising alternatives with chemical structures similar to widely used petroleum-based aromatic dicarboxylic acids. However, their use in polyester synthesis has been limited due to production challenges. Here, we report the fermentative production of five pseudoaromatic dicarboxylic acids, including 2-pyrone-4,6-dicarboxylic acid (PDC) and pyridine dicarboxylic acids (PDCAs: 2,3-, 2,4-, 2,5-, and 2,6-PDCA), from glucose using five engineered Corynebacterium glutamicum strains. A platform C. glutamicum chassis strain was constructed by modulating the expression of nine genes involved in the synthesis and degradation pathways of precursor protocatechuate (PCA) and the glucose-uptake system. Comparative transcriptome analysis of the engineered strain against wild-type C. glutamicum identified iolE (NCgl0160) as a target for PDC production. Optimized fed-batch fermentation conditions enabled the final engineered strain to produce 76.17 ± 1.24 g/L of PDC. Using this platform strain, we constructed 2,3-, 2,4-, and 2,5-PDCA-producing strains by modulating the expression of key enzymes. Additionally, we demonstrated a previously uncharacterized pathway for 2,3-PDCA biosynthesis. The engineered strains produced 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA through fed-batch fermentation. To complete the portfolio, we introduced the 2,6-PDCA biosynthetic pathway to an L-aspartate pathway-enhanced C. glutamicum strain, producing 15.01 ± 0.03 g/L of 2,6-PDCA in fed-batch fermentation. The metabolic engineering strategies developed here will be useful for the production of pseudoaromatic chemicals.

Construction and Optimization of a Yeast Cell Factory for Producing Active Unnatural Ginsenoside 3β-O-Glc2-DM.

Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3-OH and/or C20-OH of protopanaxadiol (PPD) and C6-OH and/or C20-OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc2-DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc2-DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc2-DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc2-DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc2-DM as a potential cardioprotective drug candidate.

Investigation of Bottleneck Enzyme Through Flux Balance Analysis to Improve Glycolic Acid Production in Escherichia coli.

Amid rising environmental concerns, attempts have been made to produce glycolic acid (GA) using microbial processes with renewable carbon resources instead of using chemicals. The Dahms pathway for GA production uses xylose as a substrate and consists of relatively simple enzymatic steps. However, employing it leads to a decrease in cell growth and GA productivity. Systematically identifying and addressing metabolic bottlenecks in the Dahms pathway are essential for efficient glycolic acid (GA) production have not yet been performed. Through metabolic flux balance analysis, we found that insufficient aldehyde dehydrogenase (AldA) activity lowers GA production and negatively affects cell growth due to reduced energy production. Thus, we discovered a novel AldA isolated from Buttiauxella agrestis (BaAldA) demonstrated a 1.69-fold lower KM and a 1.49-fold higher turnover rate (kcat/KM) than AldA from Escherichia coli (EcAldA). GA production in E. coli harboring BaAldA was 1.59 times higher than in the original strain. Fed-batch fermentation of E. coli harboring BaAldA produced 22.70g/L GA with a yield of 0.497g/gxylose (98.2% of the theoretical maximum yield in the Dahms pathway), showing a higher final yield for GA than previously reported in E. coli. Our novel BaAldA enzyme shows great potential for the production of GA using microorganisms or enzymes. Furthermore, our approach to identifying metabolic bottlenecks using flux balance analysis could be utilized to enhance the microbial production of various desirable products in future studies.

Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool

BackgroundGeranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis.ResultsWe first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far.ConclusionsEfficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

Metabolic engineering of Corynebacterium crenatum for enhanced L-tyrosine production from mannitol and glucose

BackgroundL-Tyrosine (L-Tyr) is a significant aromatic amino acid that is experiencing an increasing demand in the market due to its distinctive characteristics. Traditional production methods exhibit various limitations, prompting researchers to place greater emphasis on microbial synthesis as an alternative approach.ResultsHere, we developed a metabolic engineering-based method for efficient production of L-Tyr from Corynebacterium crenatum, including the elimination of competing pathways, the overexpression of aroB, aroD, and aroE, and the introduction of the mutated E. coli tyrAfbr gene for elevating L-Tyr generation. Moreover, the mtlR gene was knocked out, and the mtlD and pfkB genes were overexpressed, allowing C. crenatum to produce L-Tyr from mannitol. The L-Tyr production achieved 6.42 g/L at a glucose-to-mannitol ratio of 3:1 in a shake flask, which was 16.9% higher than that of glucose alone. Notably, the L-Tyr production of the fed-batch fermentation was elevated to 34.6 g/L, exhibiting the highest titers among those of C. glutamicum previously reported.ConclusionThe importance of this research is underscored by its pioneering application of mannitol as a carbon source for the biosynthesis of L-Tyr, as well as its examination of the influence of mannitol-associated genes in microbial metabolism. A promising platform is provided for the production of target compounds that does not compete with human food source.

Reusable Immobilized Lactobacillus futsaii CS3 for Enhanced GABA Synthesis using Low-Cost Substrates in Fermenter-Scale Batch and Fed-Batch Fermentations

On the industrial scale, the cost of commercial culture media for the production of gamma-aminobutyric acid (GABA) is a very important factor. This study employed a low-cost substrates and by-product from agri-food industry for GABA synthesis by Lact. futsaii CS3 at the fermenter-scale. Lact. futsaii CS3 cells were immobilized in 3 % (w/v) sodium alginate and employed for GABA synthesis in the optimal modified MRS medium (3.48 % (w/v) cane sugar, 3.84 % (w/v) tuna condensate waste and 10.77 % (w/v) monosodium glutamate (MSG)) with the initial pH medium of 5, fermentation temperature at 37 °C and agitation speed at 30 rpm. During the 60th h of batch fermentation without pH control, immobilized Lact. futsaii CS3 efficiently transformed MSG into the highest GABA of 19.05 g/L, achieving the volumetric productivity of 0.32 g/L/h with a bioconversion rate of 29.01 %. To further enhance GABA production, MSG (7.5 % w/v) was fed into the fermenter during the 48th h of fed-batch fermentation, aiming to amplify GABA synthesis. The maximum GABA (26.28 g/L) was synthesized at 84 h of fed-batch fermentation, and the volumetric productivity and a bioconversion rate of 0.31 g/L/h and 23.59 %, respectively, were obtained. Fed-batch fermentation significantly outperformed batch fermentation, resulting in a 37.95 % increase in GABA production. Moreover, immobilized Lact. futsaii CS3 cells could be reused in 2 batch cycles. In the 1st reusability, the maximum GABA synthesis reached 25.94 g/L, and the volumetric productivity and a bioconversion rate of 0.31 g/L/h and 23.29 %, respectively, were achieved at 84 h of fed-batch fermentation. While in the 2nd reusability, GABA synthesis decreased to 23.29 g/L, with the volumetric productivity and a bioconversion rate of 0.28 g/L/h and 20.91 %, respectively, at 84 h of fed-batch fermentation. HIGHLIGHTS GABA production utilizing agri-food industry by-product. GABA production was enhanced by cane sugar and tuna condensate waste. Immobilized futsaii CS3 produced GABA 19.05 g/L (batch). Immobilized futsaii CS3 produced GABA 26.28 g/L (fed-batch). Immobilized futsaii CS3 could be reused in 2 batch cycles on GABA production. GRAPHICAL ABSTRACT

Efficient pectin recovery from sugar beet pulp as effective bio-based coating for Pacific white shrimp preservation

This study demonstrates the valorization of sugar beet pulp (SBP)-derived pectin to produce bio-based coatings for shrimp preservation. Pectin extraction was assessed at varying temperatures and extraction times to achieve tailored properties (high methoxyl-pectins, degree of esterification-DE >79.0 %) leading to 11.5 % extraction yield, 78.1 % galactouronic acid content and 80 % DE at optimal conditions (pH 1.5, 80 °C, 2 h). Pectin-based coatings supplemented with ascorbic acid (AA) (0.5–2.0 %) led to organoleptically acceptable shrimps with significantly lower total color differences during 28-days of storage, compared to uncoated and pectin-coated counterparts. AA-based coatings delayed shrimp melanosis, expressed as reduced polyphenoloxidase activity (48–86 %). Rich-in-holocellulose solids derived after pectin extraction were used for bacterial cellulose (BC) production, pinpointing the SBP potential as a multi-purpose feedstock. Fed-batch fermentation enhanced BC concentration (by 110 %) and productivity (1.6-fold higher) compared to batch-cultures. Pectin produced within a SBP-based biorefinery could be applied as bio-based coating with food packaging potential.

Adjustment of the main biosynthesis modules to enhance the production of l-homoserine in Escherichia coli W3110.

l-homoserine is an important platform compound of many valuable products. Construction of microbial cell factory for l-homoserine production from glucose has attracted a great deal of attention. In this study, l-homoserine biosynthesis pathway was divided into three modules, the glucose uptake and upstream pathway, the downstream pathway, and the energy supply module. Metabolomics of the chassis strain HS indicated that the supply of ATP was inadequate, therefore, the energy supply module was firstly modified. By balancing the ATP supply module, the l-homoserine production increased by 66% to 12.55 g/L. Further, the results indicated that the upstream pathway was blocked, and increasing the culture temperature to 37°C could solve this problem and the l-homoserine production reached 21.38 g/L. Then, the downstream synthesis pathways were further strengthened to balance the fluxes, and the l-homoserine production reached the highest reported level of 32.55 g/L in shake flasks. Finally, fed-batch fermentation in a 5-L bioreactor was conducted, and l-homoserine production could reach to 119.96 g/L after 92 h cultivation, with the yield of 0.41 g/g glucose and productivity of 1.31 g/L/h. The study provides a well research foundation for l-homoserine production by microbial fermentation with the capacity for industrial application.

Engineering the L-tryptophan metabolism for efficient de novo biosynthesis of tryptophol in Saccharomyces cerevisiae

Tryptophol (IET) is a metabolite derived from L-tryptophan that can be isolated from plants, bacteria, and fungi and has a wide range of biological activities in living systems. Despite the fact that IET biosynthesis pathways exist naturally in living organisms, industrial-scale production of IET and its derivatives is solely based on environmentally unfriendly chemical conversion. With diminishing petroleum reserves and a significant increase in global demand in all major commercial segments, it becomes essential to develop new technologies to produce chemicals from renewable resources and under mild conditions, such as microbial fermentation. Here we characterized and engineered the less-studied L-tryptophan pathway and IET biosynthesis in the baker’s yeast Saccharomyces cerevisiae, with the goal of investigating microbial fermentation as an alternative/green strategy to produce IET. In detail, we divided the aromatic amino acids (AAAs) metabolism related to IET synthesis into the shikimate pathway, the L-tryptophan pathway, the competing L-tyrosine/L-phenylalanine pathways, and the Ehrlich pathway based on a modular engineering concept. Through stepwise engineering of these modules, we obtained a yeast mutant capable of producing IET up to 1.04 g/L through fed-batch fermentation, a ~ 650-fold improvement over the wild-type strain. Besides, our engineering process also revealed many insights about the regulation of AAAs metabolism in S. cerevisiae. Finally, during our engineering process, we also discovered yeast mutants that accumulate anthranilate and L-tryptophan, both of which are precursors of various valuable secondary metabolites from fungi and plants. These strains could be developed to the chassis for natural product biosynthesis upon introducing heterologous pathways.

Screening and stability verification of reference genes in Botrytis cinerea ZX2 fermentation

Botrytis cinerea, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´-trans-ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of TUA, TUB, ATC, EF-1, GAPDH, UCE and GTP genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the ACT was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´-trans-ABA-diol production (hmgr and bcaba3) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in B.cinerea ZX2.

Reconstitution of the Mevalonate Pathway for Improvement of Isoprenoid Production and Industrial Applicability in Escherichia coli.

Natural products, especially isoprenoids have many industrial applications, including medicine, fragrances, food additives, personal care and cosmetics, colorants, and even advanced biofuels. Recent advancements in metabolic engineering with synthetic biology and systems biology have drawn increased interest in microbial-based isoprenoid production. In order to engineer microorganisms to produce a large amount of value-added isoprenoids, great efforts have been made by employing various strategies from synthetic biology and systems biology. We also have engineered E. coli to produce various isoprenoids by targeting and engineering the isoprenoid biosynthetic pathways, methylerythritol phosphate (MEP), and mevalonate (MVA) pathways. Here, we introduced new combinations of the MVA pathway in E. coli with genes from biosafety level 1 (BSL 1) organisms. The reconstituted MVA pathway constructs (pSCS) are not only preferred to the living modified organism (LMO) regulation, but they also improved carotenoid production. In addition, the pSCS constructs resulted in enhanced lycopene production and cell-specific productivity compared to the previous MVA pathway combination (pSNA) in fed-batch fermentation. The pSCS constructs would not only bring an increase in isoprenoid production in E. coli, but they could be an efficient system to be applied for the industrial production of isoprenoids with industry-preferred genetic combinations.

Engineering the by-products pathway in Aureobasidium pullulans for highly purified polymalic acid fermentation with concurrent recovery of l-malic acid

The fermentation of polymalic acid (PMA) by Aureobasidium pullulans, followed by acid hydrolysis to release the monomer l-malic acid (l-MA), has emerged as a promising process for the bio-based production of l-MA. However, the presence of specific by-products significantly affects the quality of the final products. In this study, chassis strains harboring an overexpressed endogenous malate dehydrogenase gene (ApMDH2) were engineered to delete key genes involved in the pullulan, melanin, and liamocin biosynthetic pathways. Furthermore, to enhance PMA synthesis productivity and prevent intracellular NADPH accumulation, an irreversible trans-hydrogenase transformation system was designed to efficiently convert NADPH to NADH. In fed-batch fermentation, the engineered strain produced the highest PMA titer (194.3 ± 1.1 g/L) and l-MA yield (0.89 ± 0.01 g/g) with an increased productivity (1.45 ± 0.06 g/L∙h). Moreover, a total of 86.19 % l-MA, with a purity of 99.7 %, was successfully extracted from fermentation broth.

Biological conversion of lignin-derived ferulic acid from wheat bran into vanillin

Lignin is a promising feedstock for producing vanillin, one of the most extensively used flavor enhancers. However, the biotransformation performance of lignin derivatives into vanillin is still unsatisfactory. In this study, an efficient conversion strategy of lignin into vanillin was established by employing engineered Saccharomyces cerevisiae as a whole-cell biocatalyst. Optimization of cell culture media and whole-cell bioconversion improved the production efficiency of vanillin. The vanillin titer reached 15.3 mM with a molar yield of 71 % in fed-batch fermentation mode, while incorporating in-situ product separation, demonstrated a remarkable 2.6-fold increase. The whole-cell bioconversion, coupled with in-situ separation, successfully converted real lignin hydrolysate into a record vanillin titer of 21.1 mM, equivalent to 1.8 mg of vanillin per gram of wheat bran biomass. The whole-cell bioconversion process integrated in-situ product separation, represents a sustainable approach for vanillin production and offers a promising pathway for lignin valorization.

Microbial production of adipic acid from 6-hydroxyhexanoic acid for biocatalytic upcycling of polycaprolactone

To valorize waste polycaprolactone (PCL), one of the most widely used biodegradable plastics, into a value-added chemical, we upcycled 6-hydroxyhexanoic acid (6-HHA), the sole monomer of PCL, into adipic acid (AA) using a microbial method. Recombinant Escherichia coli strains expressing chnD (6-HHA dehydrogenase) and chnE (6-oxohexanoic acid dehydrogenase) genes from three bacteria were constructed, and all these strains successfully produced AA from 6-HHA. Among these, the E. coli strain harboring ChnDE genes from Acinetobacter strain SE19 (E. coli [pKK-AcChn]) showed the highest AA-producing ability. To increase the AA production titer, we optimized the culture temperature of this strain in flask culture and performed fed-batch fermentation in a 5 L bioreactor. After the fed-batch fermentation, the AA production titer increased to 15.6 g/L. As 6-HHA is a monomer of PCL, our results provide the groundwork for the development of a biocatalytic upcycling method of PCL.

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris.

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.

Harnessing the Plastic-degrading Potential of Pseudomonas Species for Environmental Sustainability

Abstract The prevalent utilization of synthetic plastics in contemporary society has made them indispensable. Nonetheless, the enduring predicament of plastic waste presents challenges to environmental sustainability and effective waste management. Plastic pollution can disrupt habitats and natural processes, diminishing the ecosystem’s resilience to climate change and directly impacting the livelihoods, food production capabilities, and social well-being of people. Moreover, plastic recycling itself can rise the production of polluting microplastic that find their way into water sources or air. In to address the environmental predicaments associated with plastics, it is imperative to examine the intricate interplay between microbes and polymers. Understanding this dynamic relationship is important as we strive to develop effective solutions and mitigate the adverse effects of plastic on our environment. Bacteria evolved strategies to survive and degrade plastics in contaminated environments. In this study, we focused on the ability of Pseudomonas species to degrade different sets of synthetic plastics including Low-Density polyethylene (LDPE), High-density polyethylene (HDPE), polyvinyl and polystyrene. Then a simulation study using kinetic models was conducted to determine optimum parameters for degradation ability. Our results indicate four different Pseudomonas strains P. aeruginosa strains, P. boreopolis and P. dehiensis two strains of which were able to grow in minimum media containing the different plastics as the only carbon sources reflecting their capacities to degrade these complexes polymers. Kinetic modeling for microbial growth of P. aeruginosa using batch and feed batch demonstrated that the feed batch fermentation procedure has the potential to produce Pseudomonas biomass on a larger scale, with a production of 250.7481 g/l after 72 hours. These results enhance their applicability in industrial settings particularly in bioremediation field. These findings highlight the important uses of bacterial strain of Pseudomonas as a promising strategy to depolymerize complex plastics into monomers for recycling or mineralize them into new biodegradable biomass.

Metabolic Engineering of Saccharomyces cerevisiae for Fermentative Production of Heme.

Heme is a key ingredient required to mimic the color and flavor of meat in plant-based alternatives. This study aimed to develop a yeast-based microbial cell factory for efficient and sustainable production of heme. To this end, first, Hem12p (uroporphyrinogen decarboxylase) was identified as the rate-limiting enzyme in the heme biosynthetic pathway present in Saccharomyces cerevisiae D452-2. Next, we investigated the effects of disruption of the genes involved in the competition for heme biosynthesis precursors, transcriptional repression, and heme degradation (HMX1) on heme production efficiency. Of the knock-out strains constructed in this study, only the HMX1-deficient strain produced heme at a higher concentration than the background strain without gene disruption. In addition, overexpression of PUG1 encoding a plasma membrane transporter involved in protoporphyrin IX (the precursor to heme biosynthesis) uptake led to a significant increase in intracellular heme concentration. As a result, among the various engineered strains constructed in this study, the ΔHMX1/H3&12+PUG1 strain, the HMX1-deficient strain overexpressing HEM3, HEM12, and PUG1, produced the highest concentration of heme (4.6mg/L) in batch fermentation, which was 3.9-fold higher than that produced by the wild-type D452-2 strain. In a glucose-limited fed-batch fermentation, the ΔHMX1/H3&12+PUG1 strain produced 28mg/L heme in 66h.

Fermentative production of 3-hydroxypropionic acid by using metabolically engineered Klebsiella pneumoniae strains

3-Hydroxypropionic acid (3-HP) is an industrially important platform chemical for super-absorbent or biodegradable polymers. Its production via biological methods is expected to be more competitive than chemical methods. Klebsiella pneumoniae is the most promising host due to its innate capabilities for 3-HP and vitamin-B12 production, ease of culture, and ease of engineering. In this study, step-by-step metabolic engineering and fermentation technologies were used to enhance the production of 3-HP. K. pneumoniae-derived ydcW gene was overexpressed using a plasmid after screening candidate genes. Major competing pathways encoded by dhaT, yqhD, ldhA, glpK, poxB, and pta-ackA were blocked. Additionally, it was demonstrated that simultaneous reinforcement of two native aldehyde dehydrogenase encoded by the ydcW gene preferring NADPH and the puuC gene preferring NADH, synergistically improved 3-HP production. Additional reinforcement of the acs gene to reduce acetate accumulation resulted in 93.7 g/L of 3-HP with a yield of 0.42 g/g·glycerol over a 72-h fed-batch fermentation. This performance is deemed sufficient for industrial applications.