Engineering Escherichia coli native metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.

Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli.

Vanillyl alcohol is a precursor of vanillin, which is one of the most widely used flavor compounds. Currently, vanillyl alcohol biosynthesis still encounters the problem of low efficiency. In this study, coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E. coli. First, two pathways were compared for biosynthesis of the immediate precursor 3, 4-dihydroxybenzyl alcohol in monocultures, and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway. To enhance the efficiency of the last methylation step, two strategies were used, and strengthening S-adenosylmethionine (SAM) regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect. Then, the optimized pathway was assembled in a single cell. However, the biosynthetic efficiency was still low, and was not significantly improved by modular optimization of pathway genes. Thus, coculturing engineering strategy was adopted. At the optimal inoculation ratio, the titer reached 328.9 mg/L. Further, gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain. As a result, the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation. These are the highest reported titers of vanillyl alcohol so far. This work provides an effective strategy for sustainable production of vanillyl alcohol.

Biosynthesis of plant hemostatic dencichine in Escherichia coli.

Dencichine is a plant-derived nature product that has found various pharmacological applications. Currently, its natural biosynthetic pathway is still elusive, posing challenge to its heterologous biosynthesis. In this work, we design artificial pathways through retro-biosynthesis approaches and achieve de novo production of dencichine. First, biosynthesis of the two direct precursors L-2, 3-diaminopropionate and oxalyl-CoA is achieved by screening and integrating microbial enzymes. Second, the solubility of dencichine synthase, which is the last and only plant-derived pathway enzyme, is significantly improved by introducing 28 synonymous rare codons into the codon-optimized gene to slow down its translation rate. Last, the metabolic network is systematically engineered to direct the carbon flux to dencichine production, and the final titer reaches 1.29 g L-1 with a yield of 0.28 g g-1 glycerol. This work lays the foundation for sustainable production of dencichine and represents an example of how synthetic biology can be harnessed to generate unnatural pathways to produce a desired molecule.

Targeting cofactors regeneration in methylation and hydroxylation for high level production of Ferulic acid.

Ferulic acid (FA) is a natural methylated phenolic acid which represents various bioactivities. Bioproduction of FA suffers from insufficient methyl donor supplement and inefficient hydroxylation. To overcome these hurdles, we first activate the S-adenosylmethionine (SAM) cycle in E. coli by using endogenous genes to supply sufficient methyl donor. Then, a small protein Fre is introduced into the pathway to efficiently regenerate FADH2 for the hydroxylation. Remarkably, regeneration of these two cofactors dramatically promotes FA synthesis. Together with decreasing the byproducts formation and boosting precursor supply, the titer of FA reaches 5.09 g/L under fed-batch conditions, indicating a 20-fold improvement compared with the original producing E. coli strain. This work not only establishes a promising microbial platform for industrial level production of FA and its derivatives, but also highlights a convenient and effective strategy to enhance the biosynthesis of chemicals requiring methylation and FADH2-dependent hydroxylation.

Design of stable and self-regulated microbial consortia for chemical synthesis.

Microbial coculture engineering has emerged as a promising strategy for biomanufacturing. Stability and self-regulation pose a significant challenge for the generation of intrinsically robust cocultures for large-scale applications. Here, we introduce the use of multi-metabolite cross-feeding (MMCF) to establish a close correlation between the strains and the design rules for selecting the appropriate metabolic branches. This leads to an intrinicially stable two-strain coculture where the population composition and the product titer are insensitive to the initial inoculation ratios. With an intermediate-responsive biosensor, the population of the microbial coculture is autonomously balanced to minimize intermediate accumulation. This static-dynamic strategy is extendable to three-strain cocultures, as demonstrated with de novo biosynthesis of silybin/isosilybin. This strategy is generally applicable, paving the way to the industrial application of microbial cocultures.

Co-immobilization of two-component hydroxylase monooxygenase by functionalized magnetic nanoparticles for preserving high catalytic activity and enhancing enzyme stabilty.

Cascade reactions catalyzed by two or more enzymes have been widely used in industrial production and exhibited many advantages over the single-enzyme catalytic system. In this study, two components of hydroxylase monooxygenase (HpaBC) were first co-immobilized by Ni2+-nitrilotriacetic acid (Ni-NTA) functionalized magnetic silica nanoparticles (Ni-NTA/H2N-SiO2@Fe3O4) for enhancing the stability and activity of biocatalysts with multi-components. These two components, HpaB and HpaC, were modified with histidine-tag and employed to construct a bi-enzyme catalytic system. After co-immobilization, the activity of the bi-enzyme system was 2.6 times of free enzymes. Meanwhile, the co-immobilized system was more stable against high temperature and alkaline condition, and maintained 76.6% of the initial activity for storage 12 days. Moreover, the co-immobilized HpaBC remained more than 60% catalytic activity after 7 cycles. These results showed that co-immobilized multi-component enzymes based on functionalized magnetic nanoparticles without purification would play a great potential role in the field of industrial biocatalysis.

Constructing an efficient salicylate biosynthesis platform by Escherichia coli chromosome integration.

Salicylate (SA) is an important platform chemical widely used in cosmetic and pharmaceutical industries. In this study, an efficient SA producing strain was constructed by step-by-step chromosome integration. First, the SA biosynthetic module controlled by promoters PT7 or Ptac was integrated into the chromosome of E. coli, generating the basal strain SA producing strains. PT7 performed better than Ptac and led to higher SA titers, the best of which was 233.6 ± 6.6 mg/L. Disrupting pheA/tyrA eliminated accumulation of phenylalanine and tyrosine, improving SA titer to 492.4 ± 9.3 mg/L. Strengthening the upstream pathway by enhancing expression of aroG further improved SA titer to 679.9 ± 27.1 mg/L. Furthermore, pykA/pykF were disrupted to conserve phosphoenolpyruvate, resulting in a final strain that can produce 769.8 ± 12.6 mg/L of SA using glucose and glycerol as the mixed carbon source. When using sole glycerol instead, the titer was significantly improved to 1560.6 ± 50.2 mg/L. The platform strain was further used to produce muconic acid and salicyl alcohol by introducing the downstream SA-conversion modules. In this study, we constructed an effcient SA producing E. coli strain by chromosome integration, which can be used as a platform for the production of SA derivatives.

Efficient biosynthesis of 3, 4-dihydroxyphenylacetic acid in Escherichia coli.

3, 4-Dihydroxyphenylacetic acid (3, 4-DHPA) is a phenolic acid with strong anti-oxidative activity, showing potential applications in food and pharmaceutical industries. In this study, a 3, 4-DHPA biosynthetic pathway was designed by connecting 4-hydroxyphenylacetic acid (4-HPA) biosynthesis with its hydroxylation. The starting strain produced only 46 mg/L of 4-HPA in 48 h. Enhancing the shikimate pathway increased the titer by 19-fold to 923 ± 57 mg/L. Furthermore, pykA and pykF were disrupted to conserve phosphoenolpyruvate for 4-HPA production. With this effort, 4-HPA titer was increased to 1817 ± 55 mg/L. Introducing the hydroxylase HpaBC into the 4-HPA overproducing strain resulted in 3, 4-DHPA production and the best strain produced 1856 ± 67 mg/L of 3, 4-DHPA in shake flask cultures. This work reports de novo biosynthesis of 3, 4-DHPA for the first time and provides a promising alternative for sustainable production of this valuable compound.

Establishing an Artificial Pathway for Efficient Biosynthesis of Hydroxytyrosol.

Hydroxytyrosol (HT) is a valuable natural phenolic compound with strong antioxidant activity and various physiological and pharmaceutical functions. In this study, we established an artificial pathway for HT biosynthesis. First, efficient enzymes were selected to construct a tyrosol biosynthetic pathway. Aro10 from Saccharomyces cerevisiae was shown to be a better ketoacid decarboxylase than Kivd from Lactococcus lactis for tyrosol production. While knockout of feaB significantly decreased accumulation of the byproduct 4-hydroxyphenylacetic acid, overexpression of alcohol dehydrogenase ADH6 further improved tyrosol production. The titers of tyrosol reached 1469 ± 56 mg/L from tyrosine and 620 ± 23 mg/L from simple carbon sources, respectively. The pathway was further extended for HT production by overexpressing Escherichia coli native hydroxylase HpaBC. To enhance transamination of tyrosine to 4-hydroxyphenylpyruvate, NH4Cl was removed from the culture media. To decrease oxidation of HT, ascorbic acid was added to the cell culture. To reduce the toxicity of HT, 1-dodecanol was selected as the extractant for in situ removal of HT. These efforts led to an additive increase in HT titer to 1243 ± 165 mg/L in the feeding experiment. Assembly of the full pathway resulted in 647 ± 35 mg/L of HT from simple carbon sources. This work provides a promising alternative for sustainable production of HT, which shows scale-up potential.