Metabolic Engineering Strategies Research Articles

Plant Flavonoid Production in Bacteria and Yeasts

Flavonoids, a major group of secondary metabolites in plants, are promising for use as pharmaceuticals and food supplements due to their health-promoting biological activities. Industrial flavonoid production primarily depends on isolation from plants or organic synthesis, but neither is a cost-effective or sustainable process. In contrast, recombinant microorganisms have significant potential for the cost-effective, sustainable, environmentally friendly, and selective industrial production of flavonoids, making this an attractive alternative to plant-based production or chemical synthesis. Structurally and functionally diverse flavonoids are derived from flavanones such as naringenin, pinocembrin and eriodictyol, the major basic skeletons for flavonoids, by various modifications. The establishment of flavanone-producing microorganisms can therefore be used as a platform for producing various flavonoids. This review summarizes metabolic engineering and synthetic biology strategies for the microbial production of flavanones. In addition, we describe directed evolution strategies based on recently-developed high-throughput screening technologies for the further improvement of flavanone production. We also describe recent progress in the microbial production of structurally and functionally complicated flavonoids via the flavanone modifications. Strategies based on synthetic biology will aid more sophisticated and controlled microbial production of various flavonoids.

Prospects for recycling plastic waste based on polyethylene glycol terephthalate using living systems (a review)

In recent years, the biodegradation of polyethylene glycol terephthalate has become an important direction in solving the problem of environmental pollution with plastic waste. This review generalizes the latest data on various microorganisms capable of biodegrading polyethylene glycol terephthalate. The mechanisms of enzymatic reactions of polyethylene glycol terephthalate hydrolysis and the structure of biodegradation enzymes are elucidated. Challenges to the industrial implementation of polyethylene glycol terephthalate biodegradation are considered along with proposals on the promotion of appropriate waste disposal technologies. Biodegradation comprises a promising method for the environmentally friendly and efficient disposal of waste plastics. So far, no commercial biodegradation technologies for recycling polyethylene glycol terephthalate have been developed. This area is attracting increased research attention, which is expected to result in the appearance of cost-effective and high-tech biodegradation processes. Future advances are likely to be based on synthetic biology and metabolic engineering strategies capable of constructing artificial microbial consortia and modifying microbial polyethylene glycol terephthalate hydrolases aimed at a more complete biodegradation and bioconversion of polyethylene glycol terephthalate and other complex polymers.

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone

The plant‐sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2‐pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl‐CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP‐citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl‐CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12‐ACL1‐ACC1 using fed‐batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl‐CoA‐derived polyketides from low‐cost carbon sources.

Enhanced production of hydroxy fatty acids in Arabidopsis seed through modification of multiple gene expression

BackgroundCastor (Ricinus communis L.) seeds contain unusual fatty acid, hydroxy fatty acid (HFA) used as a chemical feedstock for numerous industrial products. Castor cultivation is limited by the potent toxin ricin in its seeds and other poor agronomic traits, so it is advantageous to develop a suitable HFA-producing crop. Significant research efforts have been made to produce HFA in model Arabidopsis, but the level of HFA produced in transgenic Arabidopsis is much less than the level found in castor seeds which produce 90% HFA in seed oil.ResultsWe designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleateDelta12-hydroxylase (FAH12), diacylglycerol (DAG) acyltransferase 2 (DGAT2), phospholipid: DAG acyltransferase 1–2 (PDAT1-2), phosphatidylcholine (PC): DAG cholinephosphotransferase (PDCT) and Lyso-PC acyltransferase (LPCAT). Transgenic ArabidopsispCam5 lines produced HFA counting for 25% in seed oil. By knocking out ArabidopsisFatty acid elongase 1 (AtFAE1) in pCam5 using CRISPR/Cas9 technology, the resulted pCam5-atfae1 lines produced over 31% of HFA. Astonishingly, the pCam5-atfae1 line increased seed size, weight, and total oil per seed exceeding wild type by 40%. Seed germination, seedling growth and seed mucilage content of pCam5-atfae1 lines were not affected by the genetic modification.ConclusionsOur results provide not only insights for future research uncovering mechanisms of HFA synthesis in seed, but also metabolic engineering strategies for generating safe HFA-producing crops.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L−1 with a maximal productivity of 78 mg L−1 day−1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

Engineered Microbial Cell Factories for Sustainable Production of L-Lactic Acid: A Critical Review

With the increasing demand for the biodegradable polymer material polylactic acid and its advantage of being metabolized by the human body, L-lactic acid (L-LA) is becoming increasingly attractive in environmental protection and food industry applications. However, the supply of L-LA is not satisfied, and the price is still high. Compared to enzymatic and chemical synthesis methods, L-LA production by microbial fermentation has the advantages of low cost, large yield, simple operation, and environmental protection. This review summarizes the advances in engineering microbial cell factories to produce L-LA. First, the synthetic pathways and microorganisms for L-LA production are outlined. Then, the metabolic engineering strategies for constructing cell factories to overproduce L-LA are summarized and fermentation modes for L-LA production are also given. Finally, the challenges and prospects of the microbial production of L-LA are discussed. This review provides theoretical guidance for researchers engaged in L-LA production.

Opportunities in the microbial valorization of sugar industrial organic waste to biodegradable smart food packaging materials

Many petroleum-derived plastics, including food packaging materials are non-biodegradable and designed for single-use applications. Annually, around 175 Mt. of plastic enters the land and ocean ecosystems due to mismanagement and lack of techno economically feasible plastic waste recycling technologies. Renewable sourced, biodegradable polymer-based food packaging materials can reduce this environmental pollution. Sugar production from sugarcane or sugar beet generates organic waste streams that contain fermentable substrates, including sugars, acids, and aromatics. Microbial metabolism can be leveraged to funnel those molecules to platform chemicals or biopolymers to generate biodegradable food packaging materials that have active or sensing molecules embedded in biopolymer matrices. The smart package can real-time monitor food quality, assure health safety, and provide economic and environmental benefits. Active packaging materials display functional properties such as antimicrobial, antioxidant, and light or gas barrier. This article provides an overview of potential biodegradable smart/active polymer packages for food applications by valorizing sugar industry-generated organic waste. We highlight the potential microbial pathways and metabolic engineering strategies to biofunnel the waste carbon efficiently into the targeted platform chemicals such as lactic, succinate, muconate, and biopolymers, including polyhydroxyalkanoates, and bacterial cellulose. The obtained platform chemicals can be used to produce biodegradable polymers such as poly (butylene adipate-co-terephthalate) (PBAT) that could replace incumbent polyethylene and polypropylene food packaging materials. When nanomaterials are added, these polymers can be active/smart. The process can remarkably lower the greenhouse gas emission and energy used to produce food-packaging material via sugar industrial waste carbon relative to the petroleum-based production. The proposed green routes enable the valorization of sugar processing organic waste into biodegradable materials and enable the circular economy.

Metabolic Engineering of Saccharomyces cerevisiae for Heterologous Carnosic Acid Production.

Carnosic acid (CA), a phenolic tricyclic diterpene, has many biological effects, including anti-inflammatory, anticancer, antiobesity, and antidiabetic activities. In this study, an efficient biosynthetic pathway was constructed to produce CA in Saccharomyces cerevisiae. First, the CA precursor miltiradiene was synthesized, after which the CA production strain was constructed by integrating the genes encoding cytochrome P450 enzymes (P450s) and cytochrome P450 reductase (CPR) SmCPR. The CA titer was further increased by the coexpression of CYP76AH1 and SmCPR ∼t28SpCytb5 fusion proteins and the overexpression of different catalases to detoxify the hydrogen peroxide (H2O2). Finally, engineering of the endoplasmic reticulum and cofactor supply increased the CA titer to 24.65 mg/L in shake flasks and 75.18 mg/L in 5 L fed-batch fermentation. This study demonstrates that the ability of engineered yeast cells to synthesize CA can be improved through metabolic engineering and synthetic biology strategies, providing a theoretical basis for microbial synthesis of other diterpenoids.

Combination of protein engineering and metabolic engineering to enhance (+)‐nootkatone production in Saccharomyces cerevisiae

AbstractThe introduction of heterologous pathways into microorganisms for high‐level synthesis of valuable compounds would be extremely meaningful. However, the endogenous heterologous pathway conversion point frequently serves as a bottleneck that limits product synthesis. (+)‐Nootkatone is an important functional sesquiterpenoid with a unique grapefruit flavor, which is frequently used as a fragrance. The low catalytic efficiency of the valencene synthase CnVS, the first heterologous enzyme in the biosynthesis of (+)‐nootkatone, is the major challenge that restricts the production of (+)‐nootkatone in Saccharomyces cerevisiae. In this study, we modified CnVS on the basis of the catalytic environment and mechanism of the enzyme. The residue M560 involving in alkyl transfer during substrate cyclization was found to affect activity. By tuning the residue, mutant M560L exhibited a 60% increase in the unit cell yield of (+)‐valencene, which is the precursor of (+)‐nootkatone. Furthermore, multistep strategies of metabolic engineering were employed that transferred the metabolic flux to the target product synthesis pathway and displayed the superimposed effect of metabolic and protein engineering on (+)‐nootkatone yield. Finally, multicopy integration of tHMG1 and ERG20ERG20‐GSG‐CnVSM560L boosted the production of (+)‐nootkatone to 85.43 mg/L in flask and 804.96 mg/L (5.29 mg/[L h]) in bioreactor. In conclusion, this study presents a paradigm for constructing microbial cell factories using a combination of protein and metabolic engineering in S. cerevisiae.

Model-Guided Metabolic Rewiring for Gamma-Aminobutyric Acid and Butyrolactam Biosynthesis in Corynebacterium glutamicum ATCC13032

Simple SummaryThe fermentative production of desired chemicals from renewable resources is one of the promising biosynthetic routes to replace the petrochemical-based process. Gamma-aminobutyric acid (GABA) can be synthesized from l-glutamic acid and used as a building block for the synthesis of butyrolactam and polyamide 4 (nylon 4). The genome-scale metabolic model can predict the growth ability and metabolic flux distribution by genetic disturbances, which provides a strategy to construct a microbial cell factory for GABA and butyrolactam biosynthesis. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum ATCC13032 for GABA and butyrolactam fermentation from glucose. The biosynthetic pathways of GABA and butyrolactam were constructed by overexpressing the heterologous genes using a bi-cistronic expression cassette. The genetic modifications of the metabolic network cooperatively forced the carbon flux toward GABA and butyrolactam synthesis. This study provides new insights into engineering industrial microorganisms to produce target chemicals from renewable carbon sources.Gamma-aminobutyric acid (GABA) can be used as a bioactive component in the pharmaceutical industry and a precursor for the synthesis of butyrolactam, which functions as a monomer for the synthesis of polyamide 4 (nylon 4) with improved thermal stability and high biodegradability. The bio-based fermentation production of chemicals using microbes as a cell factory provides an alternative to replace petrochemical-based processes. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum for GABA and butyrolactam fermentation. A GABA biosynthetic pathway was constructed using a bi-cistronic expression cassette containing mutant glutamate decarboxylase. An in silico simulation showed that the increase in the flux from acetyl-CoA to α-ketoglutarate and the decrease in the flux from α-ketoglutarate to succinate drove more flux toward GABA biosynthesis. The TCA cycle was reconstructed by increasing the expression of acn and icd genes and deleting the sucCD gene. Blocking GABA catabolism and rewiring the transport system of GABA further improved GABA production. An acetyl-CoA-dependent pathway for in vivo butyrolactam biosynthesis was constructed by overexpressing act-encoding ß-alanine CoA transferase. In fed-batch fermentation, the engineered strains produced 23.07 g/L of GABA with a yield of 0.52 mol/mol from glucose and 4.58 g/L of butyrolactam. The metabolic engineering strategies can be used for genetic modification of industrial strains to produce target chemicals from α-ketoglutarate as a precursor, and the engineered strains will be useful to synthesize the bio-based monomer of polyamide 4 from renewable resources.

MICROBIAL isoprene production: an overview.

Isoprene, a volatile C5 hydrocarbon, is a precursor of synthetic rubber and an important building block for a variety of natural products, solely being produced by petrochemical routes. To mitigate the ever-increasing contribution of petrochemical industry to global warming through significant carbon (CO2) evolution, bio-based process for isoprene production using microbial cell factories have been explored. Highly efficient fermentation-based processes have been studied for little over a decade now with extensive research on the rational strain development for creating robust strains for commercial isoprene production. Most of these studies involved sugars as feedstocks and using naturally occurring isoprene pathways viz., mevalonate and methyl erythritol pathway in E. coli. Recent advances, driven by efforts in reducing environmental pollution, have focused on utilization of inorganic CO2 by cyanobacteria or syngas from waste gases by acetogens for isoprene production. This review endeavors to capture the latest relevant progress made in rational strain development, metabolic engineering and synthetic biology strategies used, challenges in fermentation process development at lab and commercial scale production of isoprene along with a future perspective pertaining to this area of research.

Engineering nonphotosynthetic carbon fixation for production of bioplastics by methanogenic archaea

The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Effects of tryptophan and phenylalanine on tryptophol production in Saccharomyces cerevisiae revealed by transcriptomic and metabolomic analyses.

Tryptophol (TOL) is a metabolic derivative of tryptophan (Trp) and shows pleiotropic effects in humans, plants and microbes. In this study, the effect of Trp and phenylalanine (Phe) on TOL production in Saccharomyces cerevisiae was determined, and a systematic interpretation of TOL accumulation was offered by transcriptomic and metabolomic analyses. Trp significantly promoted TOL production, but the output plateaued (231.02-266.31 mg/L) at Trp concentrations ≥ 0.6 g/L. In contrast, Phe reduced the stimulatory effect of Trp, which was strongly dependent on the Phe concentration. An integrated genomic, transcriptomic, and metabolomic analysis revealed that the effect of Trp and Phe on TOL production was mainly related to the transamination and decarboxylation of the Ehrlich pathway. Additionally, other genes, including thiamine regulon genes (this), the allantoin catabolic genes dal1, dal2, dal4, and the transcriptional activator gene aro80, may play important roles. These findings were partly supported by the fact that the thi4 gene was involved in TOL production, as shown by heterologous expression analysis. To the best of our knowledge, this novel biological function of thi4 in S. cerevisiae is reported here for the first time. Overall, our findings provide insights into the mechanism of TOL production, which will contribute to TOL production using metabolic engineering strategies.

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications.

Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi.

Oleaginous fungi (including fungus-like protists) are attractive in lipid production due to their short growth cycle, large biomass and high yield of lipids. Some typical oleaginous fungi including Galactomyces geotrichum, Thraustochytrids, Mortierella isabellina, and Mucor circinelloides, have been well studied for the ability to accumulate fatty acids with commercial application. Here, we review recent progress toward fermentation, extraction, of fungal fatty acids. To reduce cost of the fatty acids, fatty acid productions from raw materials were also summarized. Then, the synthesis mechanism of fatty acids was introduced. We also review recent studies of the metabolic engineering strategies have been developed as efficient tools in oleaginous fungi to overcome the biochemical limit and to improve production efficiency of the special fatty acids. It also can be predictable that metabolic engineering can further enhance biosynthesis of fatty acids and change the storage mode of fatty acids.

Metabolic Engineering and Regulation of Diol Biosynthesis from Renewable Biomass in Escherichia coli.

As bulk chemicals, diols have wide applications in many fields, such as clothing, biofuels, food, surfactant and cosmetics. The traditional chemical synthesis of diols consumes numerous non-renewable energy resources and leads to environmental pollution. Green biosynthesis has emerged as an alternative method to produce diols. Escherichia coli as an ideal microbial factory has been engineered to biosynthesize diols from carbon sources. Here, we comprehensively summarized the biosynthetic pathways of diols from renewable biomass in E. coli and discussed the metabolic-engineering strategies that could enhance the production of diols, including the optimization of biosynthetic pathways, improvement of cofactor supplementation, and reprogramming of the metabolic network. We then investigated the dynamic regulation by multiple control modules to balance the growth and production, so as to direct carbon sources for diol production. Finally, we proposed the challenges in the diol-biosynthesis process and suggested some potential methods to improve the diol-producing ability of the host.

Identification of a Novel Metabolic Target for Bioactive Triterpenoids Biosynthesis in Ganoderma lucidum.

Triterpenoids are crucial active ingredients of Ganoderma lucidum (G. lucidum) with various health benefits. Yet the low biosynthesis greatly restricts their industrial applications, novel metabolic engineering strategies are needed to further enhance Ganoderma triterpenoids production. Transcription factors play vital roles in the metabolic regulation of terpenoids, which are still scarce to study in G. lucidum. Herein, a transcription factor GlbHLH5 (GenBank No. MZ436906.1) potential for metabolic regulation of Ganoderma triterpenes was identified for the first time. MeJA could increase Ganoderma triterpenoids biosynthesis, and GlbHLH5 significantly responded to MeJA induction, suggesting GlbHLH5 is a new target for Ganoderma triterpenoids overproduction. The regulatory effect of the newly identified target was further validated by homologous gene overexpression and silence in G. lucidum. It’s demonstrated that overexpression of GlbHLH5 significantly increased triterpenoids accumulation and the key enzyme genes transcription in the biosynthetic pathway, while silencing it displayed the opposite effect, indicating GlbHLH5 could positively regulate the triterpenoids biosynthesis by activating the synergistic expression of key enzyme genes in the biosynthetic pathway. Consequently, GlbHLH5 was identified as a positive regulator and novel metabolic target for Ganoderma triterpenoids biosynthesis, it sheds new lights on the regulatory effect regulation and synthetic biology of Ganoderma triterpenoids.

Systems metabolic engineering of Streptomyces venezuelae for the enhanced production of pikromycin.

Pikromycin is an important precursor of drugs, for example, erythromycin. Hence, systems metabolic engineering for the enhanced pikromycin production can contribute to the development of pikromycin-related drugs. In this study, metabolic genes in Streptomyces venezuelae were systematically engineered for enhanced pikromycin production. For this, a genome-scale metabolic model of S. venezuelae was reconstructed and simulated, which led to the selection of 11 metabolic gene targets. These metabolic genes, including four overexpression targets and seven knockdown targets, were individually engineered first. Next, two overexpression targets and two knockdown targets were selected based on the 11 strains' production performances to engineer two to four of these genes together for the potential synergistic effects on the pikromycin production. As a result, the NM1 strain with AQF52_RS24510 (methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase) overexpression and AQF52_RS30320 (sulfite reductase) knockdown showed the best production performance among all the 22 strains constructed in this study. Fed-batch fermentation of the NM1 strain produced 295.25 mg/L of pikromycin, by far the best production titer using the native producer S. venezuelae, to the best of our knowledge. The systems metabolic engineering strategy demonstrated herein can also be applied to the overproduction of other secondary metabolites using S. venezuelae.

Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of β-Alanine in Escherichia coli.

β-Alanine, with the amino group at the β-position, is an important platform chemical that has been widely applied in pharmaceuticals and feed and food additives. However, the current modest titer and productivity, increased fermentation cost, and complicated operation are the challenges for producing β-alanine by microbial fermentation. In this study, a high-yield β-alanine-producing strain was constructed by combining metabolic engineering, protein engineering, and fed-batch bioprocess optimization strategies. First, an aspartate-α-decarboxylase from Bacillus subtilis was introduced in Escherichia coli W3110 to construct an initial β-alanine-producing strain. Production of β-alanine was obviously increased to 4.36 g/L via improving the metabolic flux and reducing carbon loss by rerouting fluxes of the central carbon metabolism. To further increase β-alanine production, mechanism-based inactivation of aspartate-α-decarboxylase was relieved by rational design to maintain the productivity at a high level in β-alanine fed-batch fermentation. Finally, fed-batch bioprocess optimization strategies were used to improve β-alanine production to 85.18 g/L with 0.24 g/g glucose yield and 1.05 g/L/h productivity in fed-batch fermentation. These strategies can be effectively used in the construction of engineered strains for β-alanine and production of its derivatives, and the final engineered strain was a valuable microbial cell factory that can be used for the industrial production of β-alanine.

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from Panax ginseng, enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titers of F2 and 3β,20S-Di-O-Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better. Furthermore, the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM, and achieving their de novo biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass, which lays a foundation for drug research and development.