Feruloyl esterase (EC 3.1.1.73) are a subclass of the carboxylic acid esterase that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls [1]. Their ability to release ferulic acid and other hydroxycinnamic acids from plant biomass makes feruloyl esterase potential biocatalysts in a wide variety of applications such as in biofuel, food and feed, pulp and paper, cosmetics, and pharmaceutical industries [[2], [3], [4]]. Bio-scouring could reduce greenhouse gas emission, ultimately reducing the carbon footprint of the scouring operation [5,6]. Feruloyl esterase is considered to be an enzyme necessary to assist lignocellulosic hydrolysis in the production of bioethanol. It is often used in combination with oxidase and hydrolase to improve the decomposition of plant materials and increase the utilization rate of fermentable carbohydrates [7]. The use of feruloyl esterase to replace chemicals in pulp bleaching can increase the brightness of the paper and reduce the pollution to the environment [8]. Adding feruloyl esterase to feed can accelerate the degradation of plant cell wall, making the feed structure more porous, thereby improving the fiber digestion of plant nutrients and animal digestion of the feed [9]. Feruloyl esterase also releases ferulic acid and p-coumaric acid while degrading lignocellulose, which has the effects of antibacterial and anti-inflammatory, promoting wound healing, anti-diabetes and scavenging free radicals, and can be used in food and medicine fields to develop functional foods and drugs [10].

The sources of feruloyl esterase can be roughly divided into plant and microbial sources. In 1999, Sancho et al. [11] detected feruloyl esterase activity in barley, and this enzyme showed significant degradation activity on four typical methyl ester and feruloylated oligosaccharides. Subsequently, the enzymatic properties of feruloyl esterase in malted finger millet [12] and maize cell walls [13] were reported successively. The feruloyl esterase in plants plays an important role in their own growth and development. Buanafina et al. [14] showed that feruloyl esterase in maize pollen may assist other endogenous enzymes by deferuloylation the stigma cell wall, thereby creating an opening for the pollen tube and promoting pollen fertilization. However, the process of identification, extraction and concentration of plant-derived feruloyl esterase is time-consuming, which greatly limits their development and utilization [13]. Microbial-derived feruloyl esterase mainly includes bacterial source and fungal source. The feruloyl esterase derived from bacterial mainly includes Clostridium, Streptomyces, Pseudomonas, and Lactobacillus, while the fungal source mainly includes Aspergillus, Penicillium, Neurospora, and Talaromyce [2,3]. Nowadays, a variety of feruloyl esterase were isolated and characterized, but the enzyme activity was generally low [15,16]. Some feruloyl esterase-producing microbial strains were mainly isolated from the intestines of humans and animals, as well as the rumen of ruminants. Most of them were anaerobic microorganisms, which were difficult to isolate and cultivate. Moreover, the enzyme activity produced was low and cannot meet the requirements of industrial applications [54]. In recent years, the feruloyl esterase derived from lactic acid bacteria (LAB) has attracted wide attention due to the safety of LAB and its wide application in food processing. Various feruloyl esterase from LAB have been identified and characterized, including Lactobacillus plantarum and Lactobacillus helveticus [17,18], and have been applied to the preparation of ferulic acid from agricultural residues. When LAB strains with feruloyl esterase activity are used for fermented food, the production of free ferulic acid is significantly increased, and its nutritional value and bioavailability of various bioactive compounds are also improved [19].

Although feruloyl esterase can be synthesized by a variety of microorganisms, it has not been directly used for large-scale production due to the limited synthesis capacity of wild strains. Due to the clear genetic background of the model strain, it is easy to use molecular biology techniques to modify the strain. Therefore, constructing genetically engineered strains to obtain stable and higher yield production strains has become a trend in feruloyl esterase biosynthesis. Several feruloyl esterase coding genes have been cloned and heterologous expressed, among which Escherichia coli, Pichia pastoris and Aspergillus niger are the most commonly used expression systems. E. coli is a classical prokaryotic gene expression host with simple cell structure, short proliferation time, simple induction method, active growth and metabolism, high expression level, and post-translational modification such as disulfide bond formation ability [20]. Compared with other host systems, E. coli is easier to operate, simpler fermentation process and more cost-effective in production. Therefore, it has become the preferred choice for the research and application of recombinant proteins [21]. As the earliest model microorganism used for recombinant protein expression, a wealth of gene expression and synthetic biology tools has been established and developed in E. coli to achieve a balance between cell growth and target product synthesis [22,23], including regulating mRNA stability [24], optimizing (5′ untranslated region) 5′ UTR structure of the operon [25], ribosome binding sites (RBS) [26], and terminator sequences [27]. By using these strategies, gene expression levels can be effectively regulated.

Considering the important role of feruloyl esterase in many industries, many studies have advanced the practical application of this enzyme preparation, but its activity and yield are still the problem to be overcome. The purpose of this study is to maximize the extracellular production of feruloyl esterase with improved activity by using the host E. coli, so as to lay the foundation for its industrial application. Our previous studies found that feruloyl esterase derived from Lactobacillus amylovorus can be stably expressed in E. coli and secreted extracellular [28,29]. In this study, the extracellular secretion of feruloyl esterase was improved through multiple strategies such as codon preference optimization, cascade T7 promoter, replacement of 5′ UTR, random mutation of N-terminal sequence, and co-expression of secretory cofactors. Subsequently, six mutants of feruloyl esterase were constructed, including Fae-T40A, Fae-Q134T, Fae-F160A, Fae-G161A, Fae-Q198A, and Fae-5 × Site. The enzymatic properties and kinetic parameters of different enzyme mutants were measured, and their substrate affinity and substrate conversion rate were compared. Since the E. coli strain used in this study can secrete feruloyl esterase, the time and money cost associated with enzyme purification process will be eliminated. Therefore, the obtained results will lay a foundation for the industrial application of feruloyl esterase, and provided a reference for the rational optimization of other enzymes.