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.

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.

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.

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.

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.

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.

Customized molecular tools to strengthen metabolic engineering of cyanobacteria

Cyanobacteria are promising oxygenic phototrophs for the production of various compounds.For their(photo)biotechnological exploitation,molecular tools are required,such as,for the introduction and expression of heterologous genes,or the modulation of enzyme activities or entire pathways.Concepts and strategies for the development of photosynthetic biomanufacturing technologies based on cyanobacteria have been extensively reviewed,as well as certain specialized aspects of their genetic manipulation.However,options for metabolic engineering of specific cyanobacterial cells are still less developed than those for other bacteria of biotechnological relevance.In addition to the standard genetic toolbox for“classical”metabolic engineering,we emphasize certain aspects,including recently developed vector systems for the extrachromosomal maintenance of genes and approaches based on clustered regularly interspaced short palindromic repeats(CRISPR)interference.We highlight the development of custom molecular tools for specific strains or products,discuss the emerging use of small regulatory proteins that appear promising for advanced metabolic engineering approaches to promote specific product formation,and provide an overview of suitable online resources.Furthermore,we discuss the current trends in this field and indicate their potential,such as using suitable product sensors that enable systematic screening,and optimization approaches.

Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production

L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.

Advances in the Plant Isoprenoid Biosynthesis Pathway and Its Metabolic Engineering

Although the cytosolic isoprenoid biosynthetic pathway, mavolonate pathway, in plants has been known for many years, a new plastidial 1–deoxyxylulose-5-phosphate (DXP) pathway was identified in the past few years and its related intermediates, enzymes, and genes have been characterized quite recently. With a deep insight into the biosynthetic pathway of isoprenoids, investigations into the metabolic engineering of isoprenoid biosynthesis have started to prosper. In the present article, recent advances in the discoveries and regulatory roles of new genes and enzymes in the plastidial isoprenoid biosynthesis pathway are reviewed and examples of the metabolic engineering of cytosolic and plastidial isoprenoids biosynthesis are discussed.

Next-Generation Plant Metabolic Engineering, Inspired by an Ancient Chinese Irrigation System

Specialized secondary metabolites serve not only to protect plants against abiotic and biotic challenges, but have also been used extensively by humans to combat diseases. Due to the great importance of medicinal plants for health, we need to find new and sustainable ways to improve the production of the specialized metabolites. In addition to direct extraction, recent progress in metabolic engineering of plants offers an alternative supply option. We argue that metabolic engineering for producing the second- ary metabolites in plants may have distinct advantages over microbial production platforms, and thus pro- pose new approaches of plant metabolic engineering, which are inspired by an ancient Chinese irrigation system. Metabolic engineering strategies work at three levels： introducing biosynthetic genes, using tran- scription factors, and improving metabolic flux including increasing the supply of precursors, energy, and reducing power. In addition, recent progress in biotechnology contributes markedly to better engineering, such as the use of specific promoters and the deletion of competing branch pathways. We propose that next-generation plant metabolic engineering will improve current engineering strategies, for the purpose of producing valuable metabolites in plants on industrial scales.

Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins (RED) production

Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g^(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.

Terpenoid Indole Alkaloids Biosynthesis and Metabolic Engineering in Catharanthus roseus

Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalicine and serpentine）. The multi-step terpenoid indole alkaloids （TIAs） biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.

Systems metabolic engineering strategies for the production of amino acids

Systems metabolic engineering is a multidisciplinary area that integrates systems biology,synthetic biology and evolutionary engineering.It is an efficient approach for strain improvement and process optimization,and has been successfully applied in the microbial production of various chemicals including amino acids.In this review,systems metabolic engineering strategies including pathwayfocused approaches,systems biology-based approaches,evolutionary approaches and their applications in two major amino acid producing microorganisms:Corynebacterium glutamicum and Escherichia coli,are summarized.

Screening neutral sites for metabolic engineering of methylotrophic yeast Ogataea polymorpha

Methylotrophic yeast Ogataea polymorpha is capable to utilize multiple carbon feedstocks especially methanol as sole carbon source and energy,making it an ideal host for bio-manufacturing.However,the lack of gene integration sites limits its systems metabolic engineering,in particular construction of genome-integrated pathway.We here screened the genomic neutral sites for gene integration without affecting cellular fitness,by genomic integration of an enhanced green fluorescent protein(eGFP)gene via CRISPR-Cas9 technique.After profiling the growth and fluorescent intensity in various media,17 genome positions were finally identified as potential neutral sites.Finally,integration of fatty alcohol synthetic pathway genes into neutral sites NS2 and NS3,enabled the production of 4.5 mg/L fatty alcohols,indicating that these neutral sites can be used for streamline metabolic engineering in O.polymorpha.We can anticipate that the neutral sites screening method described here can be easily adopted to other eukaryotes.

Metabolic engineering for the microbial production of isoprenoids:Carotenoids and isoprenoid-based biofuels

Isoprenoids are the most abundant and highly diverse group of natural products.Many isoprenoids have been used for pharmaceuticals,nutraceuticals,flavors,cosmetics,food additives and biofuels.Carotenoids and isoprenoid-based biofuels are two classes of important isoprenoids.These isoprenoids have been produced microbially through metabolic engineering and synthetic biology efforts.Herein,we briefly review the engineered biosynthetic pathways in well-characterized microbial systems for the production of carotenoids and several isoprenoid-based biofuels.