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.

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.

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.

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.

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.

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.

Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae

Metabolic engineering has been widely used for production of natural medicinal molecules.However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N~6-Methyladenosine(m^(6)A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m^(6)A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4(the yeast m^(6)A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner.Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m^(6)A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Metabolic engineering of Bacillus amyloliquefaciens for efficient production ofα-glucosidase inhibitor1-deoxynojirimycin

Owing to the feature of strongα-glucosidase inhibitory activity,1-deoxynojirimycin(1-DNJ)has broad application prospects in areas of functional food,biomedicine,etc.,and this research wants to construct an efficient strain for 1-DNJ production,basing on Bacillus amyloliquefaciens HZ-12.Firstly,using the temperature-sensitive shuttle plasmid T2(2)-Ori,gene ptsG in phosphotransferase system(PTS)was weakened by homologous recombination,and non-PTS pathway was strengthened by deleting its repressor gene iolR,and 1-DNJ yield of resultant strain HZ-S2 was increased by 4.27-fold,reached 110.72 mg/L.Then,to increase precursor fructose-6-phosphate(F-6-P)supply,phosphofructokinase was weaken,fructose phosphatase GlpX and 6-phosphate glucose isomerase Pgi were strengthened by promoter replacement,moreover,regulator gene nanR was deleted,1-DNJ yield was further increased to 267.37 mg/L by 2.41-fold.Subsequently,promoter of 1-DNJ synthetase cluster was optimized,as well as 5′-UTRs of downstream genes in synthetase cluster,and 1-DNJ produced by the final strain reached 478.62 mg/L.Last but not the least,1-DNJ yield of 1632.50 mg/L was attained in 3 L fermenter,which was the highest yield of 1-DNJ reported to date.Taken together,our results demonstrated that metabolic engineering was an effective strategy for 1-DNJ synthesis,this research laid a foundation for industrialization of functional food and drugs based on 1-DNJ.

Metabolic engineering strategies for microbial utilization of methanol

The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.

Combinatorial metabolic engineering of Saccharomyces cerevisiae for improved production of 7-dehydrocholesterol

7-Dehydrocholesterol(7-DHC),a key pharmaceutical intermediate in the production of vitamin D3,has a wide range of applications.To explore fermentative synthesis of 7-DHC,a 7-DHC-producing Saccharomyces cerevisiae strain was constructed by blocking the competitive pathway,eliminating rate-limiting steps,altering global reg-ulation,and pathway compartmentalization.After blocking the competitive pathway by disrupting ERG5 and ERG6 and introducing DHCR24 from Gallus gallus,S.cerevisiae produced 139.72 mg/L(17.04 mg/g dry cell weight,hereafter abbreviated as DCW)7-DHC.Subsequent alteration of global regulation by deleting ROX1 and overexpressing UPC2-1 increased 7-DHC production to 217.68 mg/L(37.56 mg/g DCW).To remove the accu-mulated squalene,the post-squalene pathway was strengthened by co-overexpression of PGAL1-driven ERG11 and PGAL10-driven ERG1,which improved 7-DHC titer and yield to 281.73 mg/L and 46.78 mg/g DCW,respectively,and reduced squalene content by 90.12%.We surmised that the sterol precursors in the plasma membrane and peroxisomes may not be accessible to the pathway enzymes,thus we re-localized DHCR24p and Erg2p-GGGGS-Erg3p to the plasma membrane and peroxisomes,boosting 7-DHC production to 357.53 mg/L(63.12 mg/g DCW).Iron supplementation further increased 7-DHC production to 370.68 mg/L in shake flasks and 1.56 g/L in fed-batch fermentation.This study demonstrates the power of global regulation and subcellular relocalization of key enzymes to improve 7-DHC synthesis in yeast.

Genetic tools for metabolic engineering of Pichia pastoris

The methylotrophic yeast Pichia pastoris(also known as Komagataella phaffii)is widely used as a yeast cell factory for producing heterologous proteins.Recently,it has gained attention for its potential in producing chemicals from inexpensive feedstocks,which requires efficient genetic engineering platforms.This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P.pastoris.The topics cover promoters,terminators,plasmids,genome integration sites,and genetic editing systems,with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools.Additionally,this review highlights the prospects of multiplex genome integration,fine-tuning gene expression,and single-base editing systems.Overall,the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P.pastoris.

Improvement of betanin biosynthesis in Saccharomyces cerevisiae by metabolic engineering

Betanin is a member of natural pigment betacyanins family and has extensive application in the food industry as an important natural red food colorant.Its relatively inefficient production in nature however hampers access to this phytochemicals through traditional crop-based manufacturing.Microbial bioproduction therefore represents an attractive alternative.Here,we present the construction of a Saccharomyces cerevisiae strain for betanin production.Through minimizing metabolic crosstalk,screening and modifying biosynthetic enzymes,enhancing pathway flux and optimizing fermentation conditions,a final titer of betanin of 28.7 mg/L was achieved from glucose at 25℃ in baffled shake-flask,which is the highest reported titer produced by yeast to our knowledge.This work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added betanin and other betacyanins.

Metabolic engineering strategies for microbial utilization of C1 feedstocks

The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation.Here,we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock.Accordingly,we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals.In addition,we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.

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.