Enhanced Co-culture System Using Escherichia coli and Pichia pastoris (Komagataella phaffii) for Improved Microbial Production of Valuable Plant Alkaloids

Abstract

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

INTRODUCTION

Plants produce a variety of specialized metabolites, many of which have been used as pharmaceuticals. However, a few valuable metabolites accumulate in plant cells at low levels, and their chemical syntheses are laborious tasks due to their complex structures, which makes stable supply challenging. Recently, synthetic biology has been used for the biosynthesis of specialized metabolites in microorganisms.¹⁾ Certain useful pharmaceuticals produced in Escherichia coli and yeast include artemisinic acid, the precursor of the anti-malarial drug artemisinin,²⁾ thebaine, an important opioid,^3,4) among others.¹⁾

In certain cases, however, cell growth retardation caused by extending the biosynthetic pathway resulted in low productivity in the cells. Additionally, it has been challenging to provide an optimal environment for all the enzymes involved. To circumvent these problems, modular co-culture engineering composed of multiple microorganisms has been attempted.⁵⁾ This engineering system can provide various cellular environments for the functional expression of different enzymes. For example, Saccharomyces cerevisiae expresses eukaryotic proteins, such as CYPs, because post-translational modifications and inner membrane systems exist. The use of E. coli-S. cerevisiae co-cultures for producing magnoflorine⁶⁾ and resveratrol⁷⁾ have been reported.

Recently, several studies have used the methylotrophic yeast Pichia pastoris (Komagataella phaffii) for synthetic biology.⁸⁾ Besides the basic advantage of the yeast system, P. pastoris is suitable for high cell density cultivation and abundantly produces recombinant proteins. Certain biosynthetic enzymes show higher activity in P. pastoris than in S. cerevisiae.⁹⁾ These advantages of P. pastoris have enabled the production of specialized metabolites that include resveratrol, naringenin, norcoclaurine,¹⁰⁾ catharanthine¹¹⁾ and stylopine.⁹⁾ Some metabolites, e.g., naringenin and catharanthine, are biosynthesized through the complete biosynthetic pathways.^10,11)

Although P. pastoris is a useful host,⁸⁾ constructing the entire biosynthetic pathway in a single cell remains challenging. Therefore, we examined modular co-culture engineering and previously established a novel co-culture platform for E. coli and P. pastoris.¹²⁾ In this system, we used the E. coli AN2104 strain, which produces the alkaloid intermediate reticuline,¹³⁾ and the P. pastoris B52 strain, which produces the stylopine alkaloid with anti-inflammatory activity¹⁴⁾ from reticuline,⁹⁾ as models (Supplementary Fig. S1). Our previous study showed that among several media for P. pastoris, buffered methanol-complex medium (BMMY) was appropriate for stylopine production, and a higher initial inoculation ratio of E. coli cells compared to P. pastoris cells led to stable production.¹²⁾ However, we observed the cell growth inhibition of E. coli in this co-culture.¹²⁾ Improvement of this co-culture system would result in higher production of various valuable metabolites.

RESULTS AND DISCUSSION

To examine whether P. pastoris cells negatively affect E. coli cells and reticuline production, we co-cultured E. coli cells with the original P. pastoris strain GS115. Co-culture significantly decreased reticuline production and secretion (Supplementary Figs. S2, S3), suggesting that P. pastoris cells can inhibit E. coli growth and reticuline production. Thus, we hypothesized that pre-culturing E. coli cells in BMMY medium before co-culture might improve E. coli cell growth and reticuline production, leading to higher stylopine production (Supplementary Fig. S4). To test this hypothesis, E. coli cells were cultured, and reticuline production was induced in BMMY medium for 0, 24, 36, or 48 h before co-culture (Supplementary Fig. S4). Because biosynthetic enzymes in P. pastoris were well induced and expressed after 24 h of incubation,¹²⁾ the pre-culture time for P. pastoris was set to 24 h. P. pastoris cells cultured for 24 h were inoculated at an initial optical density at 600 nm (OD₆₀₀) of 0.1 in the medium. E. coli cells were cultured each time. Higher production was observed when E. coli cells pre-cultured were used (Fig. 1, Supplementary Fig. S5). We obtained a maximum yield of 347.5 µg/L stylopine at 48 h after onset of co-culture, which was approximately 41-fold higher than the yield of 8.4 µg/L at 0 h pre-culture (Fig. 1). As the stylopine amounts in the whole culture (20 mL) in a flask, the highest amount was 37.5 µg at 96 h after onset of co-culture using E. coli cells pre-cultured for 24 h, which was 25-fold higher than the yield of 1.5 µg at 0 h pre-culture (Supplementary Fig. S5). This experiment is technically challenging in part since it requires pre-culturing both E. coli and P. pastoris to regulate growth and time. Stylopine production varies depending on each experiment, and in some cases, higher or almost equal stylopine production was observed when E. coli cells pre-cultured for 48 h were used, compared to E. coli cells pre-cultured for 24 or 36 h. Therefore, the following experiments used E. coli cells pre-cultured for 48 h.

Fig. 1. (S)-Stylopine Production from Pre-cultured E. coli and P. pastoris

Reticuline-producing E. coli cells and stylopine-producing P. pastoris cells were pre-cultured in LB medium containing antibiotics or YPD medium, respectively. Each culture was inoculated in different flasks with the BMMY medium containing IPTG (0.1 mM), glycerol (5 g/L), and antibiotics and cultured for induction. E. coli cells were pre-cultured for 0 h (a, e), 24 h (b, f), 36 h (c, g), or 48 h (d, h), and then co-culture was started by the inoculation (OD₆₀₀ of 0.1) of P. pastoris cells pre-cultured for 24 h in BMMY medium. The time-dependent production of (S)-stylopine in cells (a–d) and the medium (e–h) was determined. Results indicate the mean ± standard deviation of three experiments.

Next, we examined the initial inoculation density of P. pastoris cells (OD₆₀₀ = 0.1, 0.25, 0.5, 0.75, or 1) in the co-culture (Fig. 2, Supplementary Fig. S6). Stylopine production was observed in all cases. However, higher stylopine production was observed when P. pastoris cells were inoculated at an initial OD₆₀₀ of 0.1 (Fig. 2, Supplementary Fig. S6). A lower density of P. pastoris cells would be suitable for higher stylopine production.

Fig. 2. (S)-Stylopine Production Using Different Inoculation Ratios of P. pastoris Cells

The time-dependent production of (S)-stylopine in cells (a–e) and the medium (f–j) was determined. The pre-cultured P. pastoris cells in BMMY were collected and inoculated into flasks of E. coli cells pre-cultured for 48 h in BMMY medium with an initial OD₆₀₀ of 0.1 (a, f), 0.25 (b, g), 0.5 (c, h), 0.75 (d, i), or 1.0 (e, j). Results indicate the mean ± standard deviation of three experiments.

We looked for a different approach to enhance stylopine production. Based on the basic protocol for the Multi-Copy Pichia Expression Kit (Invitrogen, U.S.A.), an additional dose of methanol (f.c. 0.5%) every 24 h was recommended to maintain the induction of protein expression. Additional methanol supplementation into the co-culture medium (f.c. 0.5%) every 24 h significantly increased stylopine production in the medium compared to no additional methanol supplementation (Fig. 3, Supplementary Fig. S7). These results suggest that the expressions of biosynthetic enzymes in P. pastoris were maintained at high levels via methanol supplementation, which led to higher production.

Fig. 3. Induction of (S)-Stylopine Production by Methanol Supplementation

The time-dependent production of (S)-stylopine in cells (a, b) and the medium (c, d) was determined. No methanol supplementation (a, c) or 50% methanol (f.c. 0.5%) supplementation at 0 h and every 24 h (b, d). E. coli cells were pre-cultured for 48 h, and co-culture was started by the inoculation (OD₆₀₀ of 0.1) of P. pastoris cells pre-cultured for 24 h in BMMY medium. Results indicate the mean ± standard deviation of three experiments.

Feeding glycerol as the substrate enhances reticuline production in reticuline-producing E. coli cells.¹⁵⁾ To further improve the productivity of our co-culture system, 50% glycerol (f.c. 5 g/L) and 50% methanol (f.c. 0.5%) were added every 24 h. Stylopine production was significantly decreased, while higher reticuline levels remained in both cells and the medium (Supplementary Figs. S8, S9). These results suggest that glycerol has a negative effect on the enzymatic reactions of P. pastoris.

The foregoing experiments allowed us to improve the E. coli-P. pastoris co-culture system, resulting in an 80-fold improvement in the medium of stylopine over the previously used method (782.2 µg/L in induced and supplemented culture at 72 h (Fig. 3d) vs. 9.8 µg/L in 0 h-induced and non-fed culture at 72 h (Fig. 1e)). A more than 20-fold increase in the whole culture (20 mL) was also observed (32.3 µg in induced and supplemented culture at 96 h (Supplementary Fig. S7b) vs. 1.5 µg in 0 h-induced and non-fed culture cells at 96 h (Supplementary Fig. S5a)).

The results of this study are important, as P. pastoris is an attractive microorganism for the industrial production of pharmaceuticals.⁸⁾ Since reticuline is an important common intermediate for various alkaloids, such as morphine and berberine, P. pastoris cells with several biosynthetic enzymes will produce such valuable metabolites. The improved co-culture system proposed here has the potential for broader application in future studies of valuable metabolite biosynthesis and will contribute to human health and environmental protection.

Acknowledgments

We thank Ms. Yoko Nakahara (Kobe Pharmaceutical University, Japan) for assisting with the experiments. This work was supported by JSPS KAKENHI (Grant Number: 22K05433 to N.S.), Grant-in-Aid for Scientific Research on Innovative Areas (Grant Number 17H05453 to N.S.), and Nagai Memorial Research Scholarship (to M.U.) from the Pharmaceutical Society of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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