Heterologous erythromycin production across strain and plasmid construction

The establishment of erythromycin production within the heterologous host E. coli marked an accomplishment in genetic transfer capacity. Namely, over 20 genes and 50 kb of DNA was introduced to E. coli for successful heterologous biosynthetic reconstitution. However, the prospect for production levels that approach those of the native host requires the application of engineering tools associated with E. coli. In this report, metabolic and genomic engineering were implemented to improve the E. coli cellular background and the plasmid platform supporting heterologous erythromycin formation. Results include improved plasmid stability and metabolic support for biosynthetic product formation. Specifically, the new plasmid design for erythromycin formation allowed for ≥89% stability relative to current standards (20% stability). In addition, the new strain (termed LF01) designed to improve carbon flow to the erythromycin biosynthetic pathway provided a 400% improvement in titer level. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:271–276, 2018     

Optimization of a heterologous mevalonate pathway through the use of variant HMG-CoA reductases

Expression of foreign pathways often results in suboptimal performance due to unintended factors such as introduction of toxic metabolites, cofactor imbalances or poor expression of pathway components. In this study we report a 120% improvement in the production of the isoprenoid-derived sesquiterpene, amorphadiene, produced by an engineered strain of Escherichia coli developed to express the native seven-gene mevalonate pathway from Saccharomyces cerevisiae (Martin et al. 2003). This substantial improvement was made by varying only a single component of the pathway (HMG-CoA reductase) and subsequent host optimization to improve cofactor availability. We characterized and tested five variant HMG-CoA reductases obtained from publicly available genome databases with differing kinetic properties and cofactor requirements. The results of our in vitro and in vivo analyses of these enzymes implicate substrate inhibition of mevalonate kinase as an important factor in optimization of the engineered mevalonate pathway. Consequently, the NADH-dependent HMG-CoA reductase from Delftia acidovorans, which appeared to have the optimal kinetic parameters to balance HMG-CoA levels below the cellular toxicity threshold of E. coli and those of mevalonate below inhibitory concentrations for mevalonate kinase, was identified as the best producer for amorphadiene (54% improvement over the native pathway enzyme, resulting in 2.5 mM or 520 mg/L of amorphadiene after 48 h). We further enhanced performance of the strain bearing the D. acidovorans HMG-CoA reductase by increasing the intracellular levels of its preferred cofactor (NADH) using a NAD⁺-dependent formate dehydrogenase from Candida boidinii, along with formate supplementation. This resulted in an overall improvement of the system by 120% resulting in 3.5 mM or 700 mg/L amorphadiene after 48 h of fermentation. This comprehensive study incorporated analysis of several key parameters for metabolic design such as in vitro and in vivo kinetic performance of variant enzymes, intracellular levels of protein expression, in-pathway substrate inhibition and cofactor management to enable the observed improvements. These metrics may be applied to a broad range of heterologous pathways for improving the production of biologically derived compounds.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l^–1 in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

Expanding Metabolic Capabilities Using Novel Pathway Designs: Computational Tools and Case Studies

Design and selection of efficient metabolic pathways is critical for the success of metabolic engineering endeavors. Convenient pathways should not only produce the target metabolite in high yields but also are required to be thermodynamically feasible under production conditions, and to prefer efficient enzymes. To support the design and selection of such pathways, different computational approaches have been proposed for exploring the feasible pathway space under many of the above constraints. In this review, an overview of recent constraint‐based optimization frameworks for metabolic pathway prediction, as well as relevant pathway engineering case studies that highlight the importance of rational metabolic designs is presented. Despite the availability and suitability of in silico design tools for metabolic pathway engineering, scarce—although increasing—application of computational outcomes is found. Finally, challenges and limitations hindering the broad adoption and successful application of these tools in metabolic engineering projects are discussed.     

大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

细菌不饱和脂肪酸合成研究进展

脂肪酸不仅是细菌细胞膜组分，还是许多生物活性物质的合成原料。不饱和脂肪酸(unsaturated fatty acid, UFA)具有更低的相变温度，是细菌调节细胞膜流动性的重要分子，因此UFA合成途径是重要的抗菌药物筛选靶点。细菌可利用厌氧途径合成UFA，其中模式生物大肠杆菌利用经典的FabA-FabB途径合成UFA，但不同细菌中UFA合成的厌氧途径具有多样性，相关催化酶类也不尽相同；细菌还可以利用需氧途径合成UFA，利用脂肪酸脱饱和酶直接将饱和脂肪酸(saturated fatty acid, SFA)转化为不饱和脂肪酸，而不同脱饱和酶会生成不同结构的UFA，在逆境耐受、致病力等多方面发挥重要作用；细菌还可以利用单加氧酶，将脂肪酸合成途径中癸酰酰基载体蛋白(acyl carrier protein, ACP)转化为顺-3-癸烯酰ACP，并最终合成UFA。细菌脂肪酸合成相关的其他酶类在UFA合成或不同种类UFA调节中也发挥着重要作用。本文系统地总结了细菌UFA合成途径与相关酶类的多样性研究进展，旨在为进一步了解细菌UFA合成机制，并以此为靶点开发抗菌药物等方面提供理论支撑。     

Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana

Summary. Serine biosynthesis in plants proceeds by two pathways; the glycolate pathway which is associated with photorespiration and the pathway from 3-phosphoglycerate which is presumed to take place in the plastids. The 3-phosphoglycerate pathway (phosphorylated pathway) involves three enzymes catalyzing three sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and 3-phosphoserine phosphatase (PSP). cDNA and genomic clones encoding these three enzymes from spinach and Arabidopsis thaliana were isolated by means of heterologous probe screening, homologous EST clones and genetic complementation in an Escherichia coli mutant. The identity of the isolated cDNAs was confirmed by functional complementation of serine auxotrophy in E. coli mutants and/or the detection of catalytic activity in the recombinant enzymes produced in E. coli. Northern blot analyses indicated the most preferential expression of these three genes in light-grown roots. In contrast, the mRNAs of two proteins involved in the glycolate pathway (H-protein of glycine decarboxylase multienzyme complex and serine hydroxymethyltransferase) accumulated to high levels in light-grown shoots. Environmental stresses, such as high salinity, flooding and low temperature, induced changes in mRNA levels of enzymes in the plastidic phosphorylated serine biosynthetic pathway but not in that of the glycolate pathway. These results indicate that the plastidic 3-phosphoglycerate pathway plays an important role in supplying serine in non-photosynthetic tissues in plants and under environmental stresses. Received December 9, 1999 Accepted February 2, 2000     

不同致泻性大肠杆菌感染调控细胞信号通路的研究进展

腹泻性大肠杆菌是在全世界引起人类和动物疾病的主要病原之一,也给社会经济带来巨大损失。根据致病机理的不同,可将腹泻性大肠杆菌分为6种:肠致病性大肠杆菌、肠出血性大肠杆菌、肠凝集性大肠杆菌、产肠毒素大肠杆菌、扩散黏附性大肠杆菌和肠侵袭性大肠杆菌。不同致病型大肠杆菌侵入宿主的方式及引起的炎症反应有所不同。文章综合分析了致病机制不同的大肠杆菌在调控宿主细胞信号通路方式上的不同,从炎症级联反应方面阐述了不同致病类型大肠杆菌的感染特征,并探讨了炎症信号通路与病原感染、预防和治疗的关系,以期为腹泻性大肠杆菌致病机制及治疗方案的研究提供帮助。     

Front Cover Image,Volume 116, Number 12, December 2019

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 10⁷ plaque-forming units/ml and produce GFP with a yield of up to 30 μg/10⁹ colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

Abstract

This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.     

G6P-capturing molecules in the periplasm of Escherichia coli accelerate the shikimate pathway

Escherichia coli, the most studied prokaryote, is an excellent host for producing valuable chemicals from renewable resources as it is easy to manipulate genetically. Since the periplasmic environment can be easily controlled externally, elucidating how the localization of specific proteins or small molecules in the periplasm affects metabolism may lead to bioproduction development using E. coli. We investigated metabolic changes and its mechanisms occurring when specific proteins are localized to the E. coli periplasm. We found that the periplasmic localization of β-glucosidase promoted the shikimate pathway involved in the synthesis of aromatic chemicals. The periplasmic localization of other proteins with an affinity for glucose-6-phosphate (G6P), such as inactivated mutants of Pgi, Zwf, and PhoA, similarly accelerated the shikimate pathway. Our results indicate that G6P is transported from the cytoplasm to the periplasm by the glucose transporter protein EIICB^Glc, and then captured by β-glucosidase.     

Metabolic engineering strategies for butanol production in Escherichia coli

The global market of butanol is increasing due to its growing applications as solvent, flavoring agent, and chemical precursor of several other compounds. Recently, the superior properties of n-butanol as a biofuel over ethanol have stimulated even more interest. (Bio)butanol is natively produced together with ethanol and acetone by Clostridium species through acetone-butanol-ethanol fermentation, at noncompetitive, low titers compared to petrochemical production. Different butanol production pathways have been expressed in Escherichia coli, a more accessible host compared to Clostridium species, to improve butanol titers and rates. The bioproduction of butanol is here reviewed from a historical and theoretical perspective. All tested rational metabolic engineering strategies in E. coli to increase butanol titers are reviewed: manipulation of central carbon metabolism, elimination of competing pathways, cofactor balancing, development of new pathways, expression of homologous enzymes, consumption of different substrates, and molecular biology strategies. The progress in the field of metabolic modeling and pathway generation algorithms and their potential application to butanol production are also summarized here. The main goals are to gather all the strategies, evaluate the respective progress obtained, identify, and exploit the outstanding challenges.     

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides.     

The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway

Diatoms are one of the most successful groups of unicellular eukaryotic algae. Successive endosymbiotic events contributed to their flexible metabolism, making them competitive in variable aquatic habitats. Although the recently sequenced genomes of the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have provided the first insights into their metabolic organization, the current knowledge on diatom biochemistry remains fragmentary. By means of a genome‐wide approach, we developed DiatomCyc, a detailed pathway/genome database of P. tricornutum. DiatomCyc contains 286 pathways with 1719 metabolic reactions and 1613 assigned enzymes, spanning both the central and parts of the secondary metabolism of P. tricornutum. Central metabolic pathways, such as those of carbohydrates, amino acids and fatty acids, were covered. Furthermore, our understanding of the carbohydrate model in P. tricornutum was extended. In particular we highlight the discovery of a functional Entner–Doudoroff pathway, an ancient alternative for the glycolytic Embden–Meyerhof–Parnas pathway, and a putative phosphoketolase pathway, both uncommon in eukaryotes. DiatomCyc is accessible online ( http://www.diatomcyc.org ), and offers a range of software tools for the visualization and analysis of metabolic networks and ‘omics’ data. We anticipate that DiatomCyc will be key to gaining further understanding of diatom metabolism and, ultimately, will feed metabolic engineering strategies for the industrial valorization of diatoms.     

Farnesol production from Escherichia coli by harnessing the exogenous mevalonate pathway

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two‐phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway‐only control. Biotechnol. Bioeng. 2010;107: 421–429. © 2010 Wiley Periodicals, Inc.     

Improved production of adipate with Escherichia coli by reversal of β-oxidation

The linear C₆ dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

     

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.     

Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger

Background

The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.

Results

A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.

Conclusions

In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.