Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Depleting global petroleum reserves and skyrocketing prices coupled with succinct supply have been a grave concern,which needs alternative sources to conventional fuels.Oleaginous microalgae have been explored for enhanced lipid production,leading towards biodiesel production.These microalgae have short life cycles,require less labor,and space,and are easy to scale up.Triacylglycerol,the primary source of lipids needed to produce biodiesel,is accumulated by most microalgae.The article focuses on different types of oleaginous microalgae,which can be used as a feedstock to produce biodiesel.Lipid biosynthesis in microalgae occurs through fatty acid synthesis and TAG synthesis approaches.In-depth discussions are held regarding other efficient methods for enhancing fatty acid and TAG synthesis,regulating TAG biosynthesis bypass methods,blocking competing pathways,multigene approach,and genome editing.The most potential targets for gene transformation are hypothesized to be a malic enzyme and diacylglycerol acyltransferase while lowering phosphoenolpyruvate carboxylase activity is reported to be advantageous for lipid synthesis.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

Metabolic engineering of the tumor microenvironment has emerged as a new strategy.Lactate dehydrogenase A(LDHA)is a prominent target for metabolic engineering.Here,we designed a cationic lipid nanoparticle formulation for LDHA gene editing.The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein(GFP).The delivery efficiency was affected by the ratios of three components:a cationic lipid,cholesterol or its derivative,and a fusogenic lipid.The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA,yielding the lipoplex,pCas9-sgLDHA/F3.The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells.Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium.pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture.In vivo,combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice.This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

A comparative analysis of China and other countries in metabolic engineering: Output, impact and collaboration

In recent years,metabolic engineering has made great progress in both academic research and industrial applications.However,we have not found any articles that specifically analyze the current state of metabolic engineering in China in comparison with other countries.Here,we review the current development and future trends of global metabolic engineering,conduct an in-depth benchmarking analysis of the development situation of China’s metabolic engineering,and identify current problems as well as future trends.We searched publications in the Scopus database from 2015 to September 2020 in the field of metabolic engineering,and analyzed the output in general,including publication trends,research distribution,popular journals,hot topics and vital institutions,but also analyzed the share of citations,field-weighted citation impact,and production in collaboration with strategic countries in science and technology.This study aims to serve as a reference for later studies,offering a comprehensive view of China’s contribution to metabolic engineering,and as a tool for the elaboration of national public policy in science and technology.

Advancements in biocatalysis:From computational to metabolic engineering

Through several waves of technological research and un‐matched innovation strategies,bio‐catalysis has been widely used at the industrial level.Because of the value of enzymes,methods for producing value‐added compounds and industrially‐relevant fine chemicals through biological methods have been developed.A broad spectrum of numerous biochemical pathways is catalyzed by enzymes,including enzymes that have not been identified.However,low catalytic efficacy,low stability,inhibition by non‐cognate substrates,and intolerance to the harsh reaction conditions required for some chemical processes are considered as major limitations in applied bio‐catalysis.Thus,the development of green catalysts with multi‐catalytic features along with higher efficacy and induced stability are important for bio‐catalysis.Implementation of computational science with metabolic engineering,synthetic biology,and machine learning routes offers novel alternatives for engineering novel catalysts.Here,we describe the role of synthetic biology and metabolic engineering in catalysis.Machine learning algorithms for catalysis and the choice of an algorithm for predicting protein‐ligand interactions are discussed.The importance of molecular docking in predicting binding and catalytic functions is reviewed.Finally,we describe future challenges and perspectives.

Cell-free synthetic biology in the new era of enzyme engineering

With the gradual rise of enzyme engineering,it has played an essential role in synthetic biology,medicine,and biomanufacturing.However,due to the limitation of the cell membrane,the complexity of cellular metabolism,the difficulty of controlling the reaction environment,and the toxicity of some metabolic products in traditional in vivo enzyme engineering,it is usually problematic to express functional enzymes and produce a high yield of synthesized compounds.Recently,cell-free synthetic biology methods for enzyme engineering have been proposed as alternative strategies.This cell-free method has no limitation of the cell membrane and no need to maintain cell viability,and each biosynthetic pathway is highly flexible.This property makes cell-free approaches suitable for the production of valuable products such as functional enzymes and chemicals that are difficult to synthesize.This article aims to discuss the latest advances in cell-free enzyme engineering,assess the trend of this developing topical filed,and analyze its prospects.

Engineering Corynebacterium glutamicum for Geraniol Production

Geraniol is a monoterpenoid alcohol with various applications in food,cosmetics,and healthcare.Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosphate pathway.To engineer C.glutamicum to produce geraniol,two different truncated geraniol synthases (GESs) were respectively expressed,and strain LX02 expressing the truncated GESs from Valeriana officinalis (t Vo GES) produced 0.3 mg/L of geraniol.Then,three geranyl diphosphate synthases (GPPSs) were combinatorially co-expressed with t Vo GES to improve geraniol production.The amounts of produced geraniol were all higher than that produced by strain LX02.Strain LX03 co-expressing ERG20 F96W–N127W (ERG20 WW) and t Vo GES produced the highest amount,5.4 mg/L.Subsequently,the co-overexpression of1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) further increased the production to 12.2 mg/L in strain LX03.Lastly,the production of geraniol was increased to 15.2 mg/L via fermentation optimization.To our knowledge,this is the first report on the engineering of C.glutamicum to produce geraniol and thus can serve as a reference for other monoterpenoid production studies.

Serotonin enrichment of rice endosperm by metabolic engineering

In animals,serotonin is a neurotransmitter and mood regulator.In plants,serotonin functions in energy acquisition,tissue maintenance,delay of senescence,and response to biotic and abiotic stresses.In this study,we examined the effect of serotonin enrichment of rice endosperm on plant growth,endosperm development,and grain quality.To do so,TDCs and T5H were selected as targets for serotonin fortification.Overexpression of TDC1 or TDC3 increased serotonin accumulation relative to overexpression of T5H in rice grain.Transgenic lines of target genes driven by the Gt1 promoter showed better field performance than those driven by the Ubi promoter.Overexpression of T5H showed little effect on plant growth or grain physicochemical quality.In neuronal cell culture assays,serotonin induced neuroprotective action against apoptosis.Breeding of rice cultivars with high serotonin content may be beneficial for health and nutrition.

Construction of Escherichia coli by Metabolic Engineering for Synthesis of Mesaconic Acid

Mesaconic acid has a special chemical structure and can undergo a series of reactions such as polymerization and addition. It is an important chemical intermediate and widely used in material, chemical and other industries. The chemical synthesis of mesaconic acid requires nitric acid, which is dangerous and harmful to the environment. The production of mesaconic acid by microbial fermentation has the characteristics of low raw material price, high efficiency and strong specificity, and thus a strong industrial application prospect. Mesaconic acid is an intermediate product of glutamic acid degradation pathway of microorganisms such as Clostridium tetani. However, at present, few reports have been conducted on the production of mesaconic acid by metabolic engineering microorganisms. In this study, glutamate mutase(GLM) and 3-methylaspartate ammonialyase(MAL) from C. tetani were recombined and expressed in Escherichia coli, and the obtained strain, BL21(DE3)/pETDuet-1-MAL-mutS-mutE, achieved the yield of mesaconic acid of 1.06 g/L. Compared with the wild type, the yields of mesaconic acid from mutants G133A and G133S increased by 21% and 16%, respectively. After 24 h of flask fermentation, the yields of mesaconic acid reached 1.28 and 1.23 g/L, respectively. This study can provide reference for microbial synthesis of mesaconic acid.

Synthesis of betanin by expression of the core betalain biosynthetic pathway in carrot

Betalain has received increased attention because of its high nutritional value and crucial physiological functions.Based on the elucidation of its core biosynthetic pathway,betalain can be produced in additional plants by metabolic engineering.Synthesis of betalain in carrot(Daucus carota L.)can improve its nutritional quality and economic value by extracting betalain from the fleshy root,non-edible part,and processing residue of carrot.In this study,two different constructs,namely,pYB:mCD(AomelOS,BvCYP76AD1S,and BvDODA1S)and p YB:CDD(BvCYP76AD1S,BvDODA1S,and MjcDOPA5GTS),were introduced into carrot for betanin synthesis by Agrobacterium-mediated transformation.Betanin can be synthetized in both transgenic calli,and p YB:m CD-transgenic callus can be used to produce betacyanin by suspension culture.However,pYB:mCD-transgenic seedlings can synthetize betanin only by tyrosine feeding.The p YB:CDD-transgenic lines can synthetize betanin in whole plants.The betanin content in fleshy root of pYB:CDD-transgenic carrot was(63.4±9)μg·g^(-1)fresh weight according to quantitative analysis.These betanin-producing carrot plant materials can be used to synthesize betanin for industrial application or consumption as dietary sources.

Expression of a New hemA Gene from Agrobacterium radiobacter in Escherichia coli for 5-Aminolevulinate Production

A new hemA gene encoding 5-aminolevulinate (ALA) synthase was cloned from Agrobacterium ra- diobacter zju-0121. The ALA synthase catalyzes the pyridoxal phosphate-dependent condensation of succinyl coen- zyme A (succinyl-CoA) and glycine to produce ALA. Four plasmids carrying the A, radiobacter hemA gene were transformed into different E. coli strains. The effects of both genetic and physiological factors on the expression of ALA synthase and ALA production were studied. The results indicated that the final intracellular activity of ALA synthase and the production of ALA in different expression systems varied largely. Among them, the recombinant E. coli BL21 (DE3) harboring the expression plasmid pET28-A. R-hemA was the most suitable one. The effects of isopropyl-β-D-thiogalactopyranoside (IPTG) addition time, IPTG concentration, culture temperature and the initial concentration of precursors and glucose on the ALA production were also evaluated. The expressed ALA synthase accounted for about 23.7% of the intracellular soluble protein. The highest specific activity of ALA syn- thase was 13.8nmol·min-1·mg-1 of intracellular soluble protein. In the batch culture of the recombinant E. coli, the extracellular ALA concentration reached 0.9 g·L-1.

Expanding beyond canonical metabolism:Interfacing alternative elements,synthetic biology,and metabolic engineering

Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.

Recent Strategies for the Development of Biosourced-Monomers, Oligomers and Polymers-Based Materials: A Review with an Innovation and a Bigger Data Focus

After setting the ground of the quantum innovation potential of biosourced entities and outlining the inventive spectrum of adjacent technologies that can derive from those, the current review highlights, with the support of Bigger Data approaches, and a fairly large number of articles, more than 250 and 10,000 patents, the following. It covers an overview of biosourced chemicals and materials, mainly biomonomers, biooligomers and biopolymers;these are produced today in a way that allows reducing the fossil resources depletion and dependency, and obtaining environmentally-friendlier goods in a leaner energy consuming society. A process with a realistic productivity is underlined thanks to the implementation of recent and specifically effective processes where engineered microorganisms are capable to convert natural non-fossil goods, at industrial scale, into fuels and useful high-value chemicals in good yield. Those processes, further detailed, integrate: metabolic engineering involving 1) system biology, 2) synthetic biology and 3) evolutionary engineering. They enable acceptable production yield and productivity, meet the targeted chemical profiles, minimize the consumption of inputs, reduce the production of by-products and further diminish the overall operation costs. As generally admitted the properties of most natural occurring biopolymers (e.g., starch, poly (lactic acid), PHAs.) are often inferior to those of the polymers derived from petroleum;blends and composites, exhibiting improved properties, are now successfully produced. Specific attention is paid to these aspects. Then further evidence is provided to support the important potential and role of products deriving from the biomass in general. The need to enter into the era of Bigger Data, to grow and increase the awareness and multidimensional role and opportunity of biosourcing serves as a conclusion and future prospects. Although providing a large reference database, this review is largely initiatory, therefore not mimicking previous classic reviews but putting them in a multiplying synergistic prospective.

Recent advances in metabolic engineering of microorganisms for production of tyrosol and its derivatives

Tyrosol is a natural phenolic compound with antioxidant,anti-inflammatory and other biological activities,serving as an important precursor of high-value products such as hydroxytyrosol and salidroside.Therefore,the green and efficient biosynthesis of tyrosol and its derivatives has become a research hotspot in recent years.Building cell factories by metabolic engineering of microorganisms is a potential industrial production way,which has low costs and environmental friendliness.This paper introduces the biosynthesis pathway of tyrosol and presents the key regulated nodes in the de novo synthesis of tyrosol in Escherichia coli and Saccharomyces cerevisiae.In addition,this paper reviews the recent advances in metabolic engineering for the production of hydroxytyrosol and salidroside.This review can provide a reference for engineering the strains for the high-yield production of tyrosol and its derivatives.

Evolution-aided engineering of plant specialized metabolism

The evolution of new traits in living organisms occurs via the processes of mutation,recombination,genetic drift,and selection.These processes that have resulted in the immense biol ogical diversity on our planet are also being employed in metabolic engineering to optimize enzymes and pathways,create new-to-nature reactions,and synthesize complex natural products in heterologous systems.In this review,we discuss two evolution-aided strategies for metabolic engineering-directed evolution,which improves upon existing genetic templates using the evolutionary process,and combinatorial pathway reconstruction,which brings together genes evolved in different organisms into a single heterol ogous host.We discuss the general principles of these strategies,describe the technologies involved and the molecular traits they influence,provide examples of their use,and discuss the roadblocks that need to be addressed for their wider adoption.A better understanding of these strategies can provide an impetus to research on gene function discovery and biochemical evolution,which is foundational for improved metabolic engineering.These evolution-aided approaches thus have a substantial potential for improving our understanding of plant metabolism in general,for enhancing the production of plant metabolites,and in sustainable agriculture.

Engineering the microenvironment of P450s to enhance the production of diterpenoids in Saccharomyces cerevisiae

Cytochrome P450 enzymes play a crucial role as catalysts in the biosynthesis of numerous plant natural products(PNPs).Enhancing the catalytic activity of P450s in host microorganisms is essential for the efficient production of PNPs through synthetic biology.In this study,we engineered Saccharomyces cerevisiae to optimize the microenvironment for boosting the activities of P450s,including coexpression with the redox partner genes,enhancing NADPH supply,expanding the endoplasmic reticulum(ER),strengthening heme biosynthesis,and regulating iron uptake.This created a platform for the efficient production 11,20-dihydroxyferruginol,a key intermediate of the bioactive compound tanshinones.The yield was enhanced by 42.1-fold through 24 effective genetic edits.The optimized strain produced up to 67.69±1.33 mg/L 11,20-dihydroxyferruginol in shake flasks.Our work represents a promising advancement toward constructing yeast cell factories containing P450s and paves the way for microbial biosynthesis of tanshinones in the future.

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation,microbial cell factories for l-serine production using non-model microorganisms have not been reported.In this study,a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production.By blocking the degradation pathway while introducing an exporter EceamA from E.coli,l-serine titer in recombinant Z.mobilis was increased from 15.30 mg/L to 62.67 mg/L.It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway.Then,536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant,and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors.Finally,855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor.This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z.mobilis for further improvement,but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.

Plants against cancer:towards green Taxol production through pathway discovery and metabolic engineering

The diversity of plant natural products presents a rich resource for accelerating drug discovery and addressing pressing human health issues.However,the challenges in accessing and cultivating source species,as well as metabolite structural complexity,and general low abundance present considerable hurdles in developing plant-derived therapeutics.Advances in high-throughput sequencing,genome assembly,gene synthesis,analytical technologies,and synthetic biology approaches,now enable us to efficiently identify and engineer enzymes and metabolic pathways for producing natural and new-to-nature therapeutics and drug candidates.This review highlights challenges and progress in plant natural product discovery and engineering by example of recent breakthroughs in identifying the missing enzymes involved in the biosynthesis of the anti-cancer agent Taxol^(®).These enzyme resources offer new avenues for the bio-manufacture and semi-synthesis of an old blockbuster drug.

Multi-modular metabolic engineering of heme synthesis in Corynebacterium glutamicum

Heme,an iron-containing porphyrin derivative,holds great promise in fields like medicine,food production and chemicals.Here,we developed an engineered Corynebacterium glutamicum strain for efficient heme production by combining modular engineering and RBS engineering.The whole heme biosynthetic pathway was methodically divided into 5-ALA synthetic module,uroporphyrinogen III(UPG III)synthetic module and heme synthetic module for further construction and optimization.Three heme synthetic modules were compared and the siroheme-dependent(SHD)pathway was identified to be optimal in C.glutamicum for the first time.To further improve heme production,the expression of genes in UPG III synthetic module and heme synthetic module was coordinated optimized through RBS engineering,respectively.Subsequently,heme oxygenase was knocked out to reduce heme degradation.The engineered strain HS12 showed a maximum iron-containing porphyrin derivatives titer of 1592 mg/L with the extracellular secretion rate of 45.5%in fed-batch fermentation.Our study constructed a C.glutamicum chassis strain for efficient heme accumulation,which was beneficial for the advancement of efficient heme and other porphyrins production.

Multivariate modular metabolic engineering and medium optimization for vitamin B_(12)production by Escherichia coli

Vitamin B_(12)is a complex compound synthesized by microorganisms.The industrial production of vitamin B_(12)relies on specific microbial fermentation processes.E.coli has been utilized as a host for the de novo biosynthesis of vitamin B_(12),incorporating approximately 30 heterologous genes.However,a metabolic imbalance in the intricate pathway significantly limits vitamin B_(12)production.In this study,we employed multivariate modular metabolic engineering to enhance vitamin B_(12)production in E.coli by manipulating two modules comprising a total of 10 genes within the vitamin B_(12)biosynthetic pathway.These two modules were integrated into the chromosome of a chassis cell,regulated by T7,J23119,and J23106 promoters to achieve combinatorial pathway optimization.The highest vitamin B_(12)titer was attained by engineering the two modules controlled by J23119 and T7 promoters.The inclusion of yeast powder to the fermentation medium increased the vitamin B_(12)titer to 1.52 mg/L.This enhancement was attributed to the effect of yeast powder on elevating the oxygen transfer rate and augmenting the strain’s isopropyl-β-D-1-thiogalactopyranoside(IPTG)tolerance.Ultimately,vitamin B_(12)titer of 2.89 mg/L was achieved through scaled-up fermentation in a 5-liter fermenter.The strategies reported herein will expedite the development of industry-scale vitamin B_(12)production utilizing E.coli.

CRISPR-Cas9-based genome-editing technologies in engineering bacteria for the production of plant-derived terpenoids

Terpenoids are widely used as medicines,flavors,and biofuels.However,the use of these natural products is largely restricted by their low abundance in native plants.Fortunately,heterologous biosynthesis of terpenoids in microorganisms offers an alternative and sustainable approach for efficient production.Various genome-editing technologies have been developed for microbial strain construction.Clustered regularly interspaced short palin-dromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)is the most commonly used system owing to its outstanding efficiency and convenience in genome editing.In this review,the basic principles of CRISPR-Cas9 systems are briefly introduced and their applications in engineering bacteria for the production of plant-derived terpenoids are summarized.The aim of this review is to provide an overview of the current developments of CRISPR-Cas9-based genome-editing technologies in bacterial engineering,concluding with perspectives on the challenges and opportunities of these technologies.