Improving riboflavin production by knocking down ribF, purA and guaC genes using synthetic regulatory small RNA

Highlights

• The expression levels of ribF, purA and guaC genes were simultaneously regulated to increase the production of riboflavin.

• Synthetic regulatory small RNA was used for fine-tuning the expression levels of multiple genes in Escherichia coli.

• WY40 produced 1454.5mg/L riboflavin with a highest yield of 0.147g/g glucose among all reported riboflavin-producing strains.

Abstract

Riboflavin is a commercially important compound in the food, pharmaceutical, chemical, and cosmetic industries. The down-regulation of expression levels of ribF, purA and guaC genes involved in the downstream or branch reactions of riboflavin biosynthesis pathway could direct more carbon flux to riboflavin accumulation. In this study, we made an attempt to fine-tune the expression levels of the 3 genes by using synthetic regulatory small RNA to enhance riboflavin production in Escherichia coli. Firstly, each of the 3 genes was knocking down by using 5 different sRNAs, respectively, and a highest increase of 50.2 % in riboflavin titer was achieved by using anti-ribF₅ sRNA. Then this sRNA was further co-expressed with 5 anti-purA and 5 anti-guaC sRNAs to simultaneously knocking down 2 or 3 genes. Co-expression of anti-ribF₅ and anti-guaC₃ led to the highest riboflavin production of 1091.3 mg/L, which was further increased by 97.6 % compared to the base strain. Finally, the expression levels of anti-ribF₅ and anti-guaC₃ were further fine-tuned by using 4 different promoters. The best strain WY40, in which the two sRNAs were respectively expressed by P_J23100 and P_J23107 promoter, produced 1454.5 mg/L riboflavin with an increase of 163.4 % compared to the base strain. To our knowledge, it’s the first study to enhance riboflavin synthesis by simultaneously regulating the expression levels of ribF, purA and guaC genes, which led to a highest yield of 0.147 g/g glucose among all reported riboflavin-producing strains.

Introduction

Riboflavin (RF), also called vitamin B₂, is an indispensable micronutrient in living organisms, including microorganisms, plants and mammals. Riboflavin and its active forms, the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have been extensively used in the food, feed, pharmaceutical and cosmetic industries (Liu et al., 2020). Traditionally, riboflavin was synthesized and produced by chemical procedures, which had many drawbacks such as environmental pollution, high cost and high energy consumption. Therefore, the microbial fermentation method has been widely applied to RF production. The industrial RF production strains are mainly derived from the fungus Ashbya gossypii (Kato and Park, 2012) and the bacterium Bacillus subtilis (Schallmey et al., 2004). Meanwhile, other species are also engineered for RF production, including Candida famata (Dmytruk et al., 2014), Corynebacterium ammoniagenes (Koizumi et al., 2000), Pichia guilliermondii (Prokopiv et al., 2013), Eremothecium ashbyi (Sengupta et al., 2012) and Escherichia coli (Lin et al., 2014; Liu et al., 2016). A number of genetic and metabolic engineering strategies were carried out to construct riboflavin overproducing strains, which include down-regulation of flavokinase/FAD synthetase activity (Mack et al., 1998; Lin et al., 2014), overexpression of the riboflavin operon (Koizumi et al., 2000; Marx et al., 2008), enhancing the supply of precursor metabolites such as ribulose-5-phosphate and GTP (Shi et al., 2009; Wang et al., 2011; Xu et al., 2015), increasing the efficiency of energy generation (Zamboni et al., 2003; Li et al., 2006) and so on. At present, the highest titer of riboflavin is around 26–30 g/L (Lee et al., 2004; Schallmey et al., 2004), however, the yield on substrate is still far below the theoretical yield and needed to be further improved. For example, the theoretical and experimental yields of Bacillus subtilis were 0.083 and 0.010 mol/mol at a μ of 0.1 h⁻¹, respectively (Dauner and Sauer, 2001).

Several previous reports have shown that down-regulation of ribF (encoding riboflavin kinase/FMN adenylyltransferase) and purA (encoding adenylosuccinate synthetase) genes was effective on riboflavin synthesis or enhancing GTP supply, as they involved in the downstream or branch reactions of riboflavin biosynthesis pathway (Fig.S1). For instance, knocking down ribF gene in Escherichia coli by changing its RBS effectively increased the production of riboflavin by 77.0 % (Lin et al., 2014). A mutant PurA with deceased catalysis activity was used to reduce the flux from IMP to AMP in Bacillus subtilis, resulting in an increase of 40.2 % in riboflavin titer (Xu et al., 2015). What’s more, knocking out guaC gene (encoding GMP reductase) in Bacillus subtilis blocked the flux from GMP to IMP and showed a 0.51-fold increase in GTP concentration (Deng et al., 2019), which indicates guaC knockdown has potential role in riboflavin accumulation. However, there are few studies on the synergistic regulation of ribF, purA and guaC genes for enhancing riboflavin production. Recently, a general strategy for simultaneously fine-tuning the expression levels of multiple genes using synthetic regulatory small RNA (sRNA, Fig. S2) has been reported (Na et al., 2013; Yoo et al., 2013). Owing to its easy implementation and does not require construction of strain libraries, synthetic sRNA has been wildly applied for metabolic engineering of Escherichia coli, Shewanella oneidensis MR-1, Bacillus subtilis, Clostridium acetobutylicum and Corynebacterium glutamicum (Liu et al., 2014; Cao et al., 2017; Cho and Lee, 2017; Sun et al., 2019; Xie et al., 2021), which successfully optimized the production of Tyrosine, Phenol, 1,3-Diaminopropane, Malonyl-CoA and Immunoglobulin G (Kim et al., 2014; Chae et al., 2015; Kim et al., 2015; Sun et al., 2018; Zhang et al., 2020).

The ribF gene is essential in Escherichia coli MG1655 (Gerdes et al., 2003), and the purA knockout strain could not grow in minimal medium without adenine addition (Hoffman et al., 2001; Sleiman et al., 2021). Besides, guaC deletion in WY0T, a riboflavin-producing base strain, resulted in severely impaired cell growth (data not shown), which might be due to the imbalance between AMP and GMP biosynthesis resulted from completely blocking the formation of IMP from GMP (Mager and Magasanik, 1960; Roberts et al., 1988). Therefore, in this study, the expression levels of these 3 genes were down-regulated by using synthetic sRNA, and the corresponding effect on riboflavin synthesis was tested in WY0T. We first designed 15 synthetic sRNAs to regulate the expression levels of these 3 genes. Anti-ribF₅ sRNA was then combined with other sRNAs to further increase riboflavin production. Finally, the expression levels of anti-ribF₅ and anti-guaC₃ were fine-tuned by using 4 different promoters. By doing so, the best strain WY40 produced 1454.5 mg/L riboflavin with a yield of 0.147 g/g glucose, which was 163.4 % and 162.5 % higher than that obtained with WY0T.