Distributing a metabolic pathway among a microbial consortium enhances production of natural products

Abstract

Metabolic engineering of microorganisms such as Escherichia coli and Saccharomyces cerevisiae to produce high-value natural metabolites is often done through functional reconstitution of long metabolic pathways. Problems arise when parts of pathways require specialized environments or compartments for optimal function. Here we solve this problem through co-culture of engineered organisms, each of which contains the part of the pathway that it is best suited to hosting. In one example, we divided the synthetic pathway for the acetylated diol paclitaxel precursor into two modules, expressed in either S. cerevisiae or E. coli, neither of which can produce the paclitaxel precursor on their own. Stable co-culture in the same bioreactor was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast. This synthetic consortium produced 33 mg/L oxygenated taxanes, including a monoacetylated dioxygenated taxane. The same method was also used to produce tanshinone precursors and functionalized sesquiterpenes.

Figure 1

A competitive E. coli – S. cerevisiae consortium for production of oxygenated taxanes. (a) Both E. coli TaxE1 and the yeast TaxS1 grew on glucose; E. coli TaxE1 produced taxadiene which can diffuse to the yeast, where it is oxygenated. (b) Only the co-culture produced the oxygenated taxanes. (c) Growth of E. coli TaxE1 was inhibited by the presence of the yeast. (d) The taxane productivity of E. coli TaxE1 was compromised by the presence of the yeast. Total taxanes = Taxadiene + Oxygenated taxanes. (e) These inhibitions could be due to the ethanol produced by the yeast, which was confirmed by follow-up experiments (Supplementary Fig. 2). Error bars, s.e. in all graphs (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle), which indicates the number of replicates for each experiment.

Figure 2

A mutualistic E. coli – S. cerevisiae consortium for production of oxygenated taxanes. (a) E. coli TaxE1 grew on xylose and produced acetate that served as sole carbon source for the yeast to grow. The taxadiene produced by E. coli TaxE1 was oxygenated in yeast TaxS1. (b) Yeast TaxS1 could only grow in presence of the E. coli TaxE1. (c) Yeast TaxS1 removed the acetate produced by E. coli TaxE1. (d) The presence of yeast TaxS1 did not compromise taxane production of E. coli TaxE1. (e) Yeast TaxS1 can only produce oxygenated taxanes when E. coli TaxE1 supplied taxadiene. The taxadiene oxygenation efficiency of this co-culture was 8% (4 mg/L out of 50 mg/L taxadiene was oxygenated). Error bars, s.e. in all graphs (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle), which indicates the number of replicates for each experiment.

Figure 3

Optimizing the yeast growth and engineering the yeast promoters improved production of the oxygenated taxanes. (a) Growth optimization (increasing the yeast inoculum and feeding additional nutrients) improved production of the oxygenated taxanes by more than two-fold. (b) A stronger promoter (UAS-GPDp), compared to the previously used TEFp, was found in the promoter screening in terms of taxadiene oxygenation. (c) The co-culture using UAS-GPDp also produced significantly (p<0.01, based on Student’s t-test) more oxygenated taxanes than that using TEFp. Error bars, s.e. in all graphs (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle), which indicates the number of replicates for each experiment.

Figure 4

Inactivating oxidative phosphorylation of the E. coli improved yeast growth and production of the oxygenated taxanes. (a) Inactivation of the E. coli oxidative phosphorylation forces the production of acetate, which became the major pathway of generating ATP in the E. coli. (b) The acetate-overproducing E. coli (TaxE4) improved the yeast growth in the co-culture. Control: TaxE1-TaxS4 co-culture; Knockout: TaxE4-TaxS4 co-culture. (c) The taxadiene oxygenation efficiency was greatly improved when the S. cerevisiae was co-cultured with the acetate-overproducing E. coli. Oxygenation efficiency of the TaxE1-TaxS4 co-culture was ~50% (20 mg/L oxygenated taxanes per 40 mg/L total taxanes), and that of the TaxE4-TaxS4 co-culture was ~75% (30 mg/L oxygenated taxanes per 40 mg/L total taxanes). Error bars, s.e. in all graphs (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle, except b, in which N=4), which indicates the number of replicates for each experiment.

Figure 5

Production of a monoacetylated dioxygenated taxane by the E. coli – S. cerevisiae co-culture. (a) Early paclitaxel biosynthetic pathway. (b) The yeast co-expressing 5αCYP-CPR, TAT and 10βCYP-CPR (TaxS6) produced putative taxadien-5α-acetate-10β-ol when co-cultured with a taxadiene-producing E. coli. Extracted ion chromatograms (346 m/z, molecular weight of monoacetylated dioxygenated taxane) are shown here. 5αCYP: TaxE4/TaxS4 co-culture; 5αCYP-TAT-10βCYP: TaxE4/TaxS6 co-culture. (c) Using a stronger promoter (UASGPDp) to express TAT improved titer of the monoacetylated dioxygenated taxane. Operating the bioreactor at a carbon-limited (CL) condition further improved the production titer and yield (xylose consumption was reduced by 30%). TEFp-TAT: TaxE4/TaxS6 co-culture, where expression of TAT was driven by TEFp; UASGPDp-TAT: TaxE4/TaxS7 co-culture, where UASGPDp was used to express TAT; UASGPDp-TAT CL: TaxE4/TaxS7 co-culture at a carbon limited condition. Error bars, s.e. in all graphs (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle), which indicates the number of replicates for each experiment.

Figure 6

Use of the E. coli, S. cerevisiae co-culture for production of other oxygenated Isoprenoids. (a) Illustration of biosynthetic pathways of ferruginol and nootkatone. (b) An E. coli was engineered to produce miltiradiene from xylose (TaxE7), which cannot produce ferruginol on its own. When this E. coli was co-cultured with a yeast expressing a specific CYP and its reductase (TaxS8), the co-culture can produce 18 mg/L ferruginol. Mass spectrum of the produced ferruginol was identical to the one in the literature (data not shown). (c) Similarly, an E. coli was engineered to produce valencene (TaxE8); itself cannot produce any oxygenated valencene. When it was co-cultured with a yeast expressing a specific CYP and its reductase (TaxS9), the co-culture can produce 30 mg/L nootkatol and low quantity of nootkatone. When an alcohol dehydrogenase was introduced to TaxS9, the resulting strain TaxS10 can produce 4 mg/L nootkatone in presence of TaxE8. Error bars, s.e. in all graphs. (some error bars are smaller than the plot symbols). All replicates have also been plotted in all graphs (open circle), which indicates the number of replicates for each experiment.