First report on Vitamin B9 production including quantitative analysis of its vitamers in the yeast Scheffersomyces stipitis

Abstract

Background The demand for naturally derived products is continuously growing. Nutraceuticals such as pre- and post-biotics, antioxidants and vitamins are prominent examples in this scenario, but many of them are mainly produced by chemical synthesis. The global folate market is expected to register a CAGR of 5.3% from 2019 to 2024 and reach USD 1.02 billion by the end of 2024. Vitamin B9, commonly known as folate, is an essential micronutrient for humans. Acting as a cofactor in one-carbon transfer reactions, it is involved in many biochemical pathways, among which the synthesis of nucleotides and amino acids. In addition to plants, many microorganisms can naturally produce it, and this can pave the way for establishing production processes. In this work, we explored the use of Scheffersomyces stipitis for the production of natural vitamin B9 by microbial fermentation as a sustainable alternative to chemical synthesis. Results Glucose and xylose are the main sugars released during the pretreatment and hydrolysis processes of several residual lignocellulosic biomasses (such as corn stover, wheat straw or bagasse). We optimized the growth conditions in minimal medium formulated with these sugars and investigated the key role of oxygenation and nitrogen source on folate production. Vitamin B9 production was first assessed in shake flasks and then in bioreactor, obtaining a folate production up to 3.7 ± 0.07 mg/L, which to date is the highest found in literature when considering wild type microorganisms. Moreover, the production of folate was almost entirely shifted toward reduced vitamers, which are those metabolically active for humans. Conclusions For the first time, the non-Saccharomyces yeast S. stipitis was used to produce folate. The results confirm its potential as a microbial cell factory for folate production, which can be also improved both by genetic engineering strategies and by fine-tuning the fermentation conditions and nutrient requirements.

Scheffersomyces stipitis ability to valorize different residual biomasses for vitamin B9 production

“…Growth in UGM 5% + 4.6 g/L urea +1 g/L MgSO 4 + 6 g/L KH 2 PO 4 was assessed in shake flasks (Figure 4C ): the cells reached 17 OD, doubling the value obtained in UGM 5%, and comparable to the biomass reached in kinetics with (NH 4 ) 2 SO 4 and salts. Glucose and fructose are both depleted and, coherently to the proposed mechanism of urea uptake (Mastella et al, 2022 ), an increase in pH at the end of the fermentation (from 5.5 to 7.5–8) is observed. Due to the faster growth, aeration becomes insufficient, as the production of 2.5 g/L ethanol is observed.…”

“…The addition of MgSO 4 and KH 2 PO 4 did not significantly ameliorate the growth (compare with Figure 4B ); the addition of (NH 4 ) 2 SO 4 as nitrogen source – either alone or in combination with MgSO 4 and KH 2 PO 4 – allowed to reach a higher biomass titre, with a faster and better consumption of sugars if compared with what observed on the sole UGM 5%; however, a very long lag phase (>72 h) was observed, and the pH at the end of the fermentation was low (pH 2.5–3). This could be explained by the mechanism of ammonium uptake, which requires the pumping of a proton in the medium (Cueto‐Rojas et al, 2017 ; Mastella et al, 2022 ). These results suggest that nitrogen is a limiting factor, but that (NH 4 ) 2 SO 4 is not the ideal source for S. stipitis on UGM 5%; moreover, the addition of MgSO 4 and KH 2 PO 4 in combination with (NH 4 ) 2 SO 4 allowed to reach a higher biomass titre and the complete depletion of both glucose and fructose, suggesting that these two salts are also a limiting factor.…”

“…We then optimized the growth and sugar consumption of S. stipitis using the three different biomasses. In particular, we focused on inoculum pre‐adaptation, oxygenation and the addition of key nutrients for supporting growth based on the experience from our previous study on defined media (Mastella et al, 2022 ).…”