Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound, widely occurring in plants, and the precursor of other natural products, such as chlorogenic acid, rosmarinic acid and caffeic acid phenethyl ester (Wu et al., 2011; David et al., 2015). Moreover, caffeic acid has numerous applications in functional foods, natural health products and medicine, because of its biological properties, which include anti-oxidant (Son and Lewis, 2002), anti-virus (Ikeda et al., 2011), anti-cancer (Espindola et al., 2019), and anti-inflammatory activities (Chao et al., 2009). Although caffeic acid can be produced either by extraction from plants or by chemical synthesis, both approaches have drawbacks, such as complex separation processes, the production of organic wastes and high cost (Yoshimoto et al., 2005). With the development of synthetic biology and metabolic engineering, heterologous biosynthesis of caffeic acid in microorganisms (e.g., Escherichia coli, Saccharomyces cerevisiae) provides a potential alternative source (Cao et al., 2020).

As the precursors of caffeic acid in the plant phenylpropanoid pathway, aromatic amino acids (AAA; i.e., L-phenylalanine, L-tyrosine) can be produced by microorganisms through the shikimic acid pathway (Krivoruchko and Nielsen, 2015; Figure 1). There has been considerable research effort applied to relieving retro-inhibition and redirecting carbon flux from glucose into AAA metabolism to maximize the pool of L-tyrosine (Tyr) and L-phenylalanine (Phe) (Huccetogullari et al., 2019; Liu Q. et al., 2019). In plants, phenylalanine ammonia lyase (PAL) and two cytochrome P450 monooxygenases, cinnamate-4-hydroxylase (C4H) and p-coumarate 3-hydroxylase (C3H) participate in the synthesis of caffeic acid from L- Phe (Kim et al., 2011; Figure 1). Alternatively, p-coumaric acid (p-CA), the direct precursor of caffeic acid in plants, can be generated by microbial tyrosine ammonia lyase (TAL) from L-Tyr directly, instead of by C4H and PAL from L-Phe (Hernandez-Chavez et al., 2019). In addition, new enzymes in heterologous bacterial hosts have been investigated as alternatives for hydroxylation of p-CA to synthesize caffeic acid. Berner et al. (2006) introduced the C3H encoded by sam5 from Saccharothrix espanaensis and Furuya et al. (2012) used the cytochrome P450 monooxygenases CYP199A2 from Rhodopseudomonas palustris. Lin and Yan (2012) found it is difficult to express the plant-originated P450 enzymes in prokaryotic systems, and identified that an E. coli endogenous non-P450 hydroxylase complex 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H, also named HpaBC) was capable of converting p-CA to caffeic acid efficiently. Huang et al. (2013) reported the de novo production of caffeic acid (766.68 mg/L) by overexpressing the E. coli endogenous HpaBC. Jones et al. (2017) achieved the highest caffeic acid titer (1.03 g/L) from a simple carbon source using EcHpaBC combined with optimization of culture conditions.

S. cerevisiae is widely used as a host organism for the biosynthesis of plant-derived natural products and has GRAS (generally recognized as safe) status (Borodina and Nielsen, 2014). The first report on caffeic acid production (289.4 mg/L) in S. cerevisiae, using HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica depended on supplementation with tyrosine (500 mg/L), the cost of which is too high for industrial production (Liu L. et al., 2019). Li et al. (2020) reported the de novo biosynthesis of caffeic acid (11.4 mg/L) from glucose, by simultaneous expression of RcTAL and C3H, assisted by cytochrome P450 reductase 1 (CPR1) in S. cerevisiae, without the need for precursor supplementation. In these two studies, they used the plasmid-based episomal expression method, at least two incompatible plasmids with diverse auxotrophic markers were applied to express caffeic acid. Recently, Zhou et al. (2021) reported the genomic integration of the caffeic acid synthetic genes RgTAL from Rhodotorula glutinis, HpaB from P. aeruginosa and HpaC from S. enterica, under the regulation of a modified GAL system and obtained the highest titer so far of caffeic acid (569.0 mg/L) in S. cerevisiae.

Compared with plasmid-based expression methods, multi-copy chromosomal integration offers advantages such as genetic regulation, passage stability, maintenance of target genes and less metabolic burden for the expression (Shi et al., 2014), and has not been applied for caffeic acid biosynthesis in yeast. Besides, in many cases, microbial heterologous synthesis of target products may be affected by a restricted copy number (Ko et al., 2020), so multi-copy integration (such as delta and rDNA integration) may be more suitable for heterologous synthesis (Malci et al., 2020) than single-copy site integration. In yeast, delta sites are located in the transposon Ty of the yeast genome and there are over 400 delta sites in S. cerevisiae (Lee and Da Silva, 1997). Genomic integration based on delta sites has been applied to the production of commercially valuable compounds by engineered S. cerevisiae, including resveratrol (Liu et al., 2017), glucaric acid (Chen et al., 2018), 2,3-butanediol (Yamada et al., 2017), and carotenoids (Yamada et al., 2018).

The ribosomal DNA (rDNA) sequence refers to the DNA sequence in the nucleus that encodes ribosomal RNA, which contains 100–140 repeat units in the S. cerevisiae XII chromosome (Kobayashi and Ganley, 2005). Each rDNA unit consists of two transcriptional regions (5S- and 35S-rDNA) and two non-transcribed spacers (NTS1 and NTS2) (Kobayashi et al., 2004). There have been many reports on the synthesis of secondary metabolic products using rDNA sites in S. cerevisiae, such as ginsenosides (Dai et al., 2013), glycyrrhetinic acid (Zhu et al., 2018), lycopene (Li et al., 2019), and (2S)-naringenin (Li et al., 2021). Thus, the multi-copy integration of heterologous pathway genes into S. cerevisiae may have great potential for the production of AAA-derived compounds like caffeic acid.

Previous studies have shown that deletion of the TPI1 gene encoding triose phosphate isomerase from glycolytic pathway adversely impacts yeast cell growth with glucose as the sole carbon source (Compagno et al., 1999, 2001). Recently, Zeng et al. (2021) increased the endogenous 2 μ plasmid viability and plasmid copy number by moving the essential gene TPI1 from chromosome to 2 μ plasmid, which provided an improved expression system in yeast. POT1, an essential gene from Schizosaccharomyces pombe, also encodes triose phosphate isomerase, which may struggle to function well in foreign host cells such as S. cerevisiae and therefore necessitate higher copy numbers in integration sites to compensate for the defective TPI1 function (Hou et al., 2012). Thus, POT1 could be used as a selection marker, making it possible to select recombinant S. cerevisiae integrated strains with higher gene copy number and stable production of heterologous products in non-selective medium such as YPD.

Herein, multi-copy integration expression strategies were applied and compared in producing caffeic acid heterologously from glucose using S. cerevisiae, providing fundamental insights to facilitate future research in this area.

In this study, the valuable product caffeic acid was heterologously synthesized through several strategies in S. cerevisiae (Figure 1), laying a foundation for further engineering of microbial cell factories. There was concern that using more than two incompatible plasmids from the same incompatibility group to carry genes expressing caffeic acid biosynthetic enzymes would lead to plasmid instability (Lindsey et al., 2011; Wang et al., 2021). Therefore, three gene cassettes were assembled into one plasmid skeleton to overcome plasmid incompatibility. Strains NKP4-6 outperformed NKP1-3, in terms of caffeic acid production, indicating that NKC6 provided more endogenous tyrosine for downstream heterologous synthesis of p-CA and caffeic acid, than the wild-type BY4742 strain, which is consistent with the report of Li et al. (2020) But the caffeic acid titers of NKP4-6 were still low (0.3 mg/L).

Huang et al. (2013) proved more p-CA supply would increase the production of caffeic acid. In the constructed caffeic acid biosynthesis pathway, p-CA serves as the direct precursor of caffeic acid. The supply of precursors is an important factor affecting the yield of products in microorganisms, but adding external precursors is likely to be problematic because exogenous compounds may adversely impact glucose metabolism (Du et al., 2011; Zhang and Hong, 2020). Thus, to be independent of external precursor supplementation, we enhanced the supply of precursor p-CA by employing high-copy coTAL integrated strain QT3-20, which can grow in YPD medium without selection pressure and provide stable p-CA precursor for the subsequent synthesis of caffeic acid. The caffeic acid titer of NKP7 haboring plasmid pLC-c3 was remarkably higher than that of NKP4-6 in both selective Sc-His and rich MYPD media, stating that expanding p-CA supply by QT3-20 did significantly increase caffeic acid production.

Previous studies showed that plasmid instability hinders the stable expression of exogenous genes and that expressing multiple biosynthetic pathway genes in a single copy usually cannot meet the need for high expression (Malci et al., 2020; Li et al., 2021). Therefore, multi-copy random chromosomal integration at multiple loci across the genome may be a superior alternative to produce high-yielding strains with optimal copy numbers of foreign genes (Da Silva and Srikrishnan, 2012). In this design, delta and rDNA clusters were selected as integration sites. Among the hundreds of potential integration sites, only a few copies of coC3H and coCPR1 were integrated into the QT3-20 genome. In general, the copy numbers of coC3H and coCPR1 from rDNA integration were higher than those from delta integration, possibly because the length of NTS sites being much longer than that of delta sites; and the use of longer homology arms improves homologous recombination frequency in S. cerevisiae (Storici et al., 2003; Maervoet et al., 2014; Juhas and Ajioka, 2016). In addition, cocktail integration and single step integration were also performed, but the results were not as good as the two-steps integration (data not shown), which may be because coTAL and coC3H encode enzymes with different functions and arrange sequentially in the caffeic acid synthetic pathway.

Recent research indicates that gene copy number influences transcription levels, thereby affecting the production efficiency of heterologous proteins (Maury et al., 2016). Although there was no significant difference in coC3H and coCPR1 transcription levels between all delta and rDNA integrated strains, the ratio of overall transcription level to gene copy number of delta-integrated strains was significantly higher than that of rDNA-integrated strains, indicating that delta integration is superior to rDNA integration in expression efficiency for caffeic acid heterologous biosynthesis. In S. cerevisiae, delta sites are dispersed across different chromosomes, whereas rDNA clusters are only found in chromosome XII; the intergenic regions are responsible for maintaining the stability of the nucleolus and stabilizing the rDNA repeat numbers (Cahyani et al., 2015). The disruption of even a few rDNA repeats among the hundreds of copies can affect cell viability, and hinder growth and development, because the intergenic region NTS1 contains an origin of DNA replication (rARS), whereas NTS2 contains both a bidirectional RNA polymerase II-dependent promoter and a replication fork barrier (RFB) (Kwan et al., 2013; Kobayashi and Sasaki, 2017). To see whether it affects translation, the total protein levels of the controlled strains NKC6, QT3-20 and engineered strains N15, D9 were measured (Supplementary Figure 2). There was no significant difference in the proportion of the wet weight (BCA colorimetry) or dry weight (Kjeldahl method) of the protein between these strains in the stable phase, showing that occupying less than 10% integration sites didn’t affect the translation and growth of integrated strains.

Caffeic acid production by D9 increased 50-fold, from 0.3 mg/L to 15.1 mg/L, after optimization, compared with the initial 2 μ multi-copy plasmid construct. The final caffeic acid titer of D9 was also 32% higher than in a previous report, by Li et al. (2020) (11.4 mg/L). Cytochrome P450 monooxygenase C3H is the rate-limiting enzyme in the caffeic acid biosynthetic pathway, coC3H copy number can be further increased, while the limited integration efficiency of exogenous biosynthetic pathways into S. cerevisiae needs to be improved. Shi et al. (2016) introduced double-stranded breaks (DSBs) in the delta sequences using the CRISPR-Cas system and substantially improved integration efficiency, which provides a potential strategy to further improve caffeic acid titers. Besides, NAD(P)H was required for catalyzing hydroxylation reactions by cytochrome P450s as a cofactor (Jung et al., 2011; Bernhardt and Urlacher, 2014; Xu et al., 2019), thus playing an essential role in involving in the p-CA hydroxylation reaction by C3H. Therefore, increasing the availability of NAD(P)H may improve C3H expression and its enzymatic activity.

In conclusion, multi-copy strategies were employed in this work to heterologously manufacture caffeic acid from glucose in S. cerevisiae, the growth, titer, gene copy number, transcription level, total protein level, and strain robustness of the engineered integrated strains were characterized carefully, resulting in a 50-fold increase in caffeic acid biosynthesis without altering the strain’s genetic stability. Through the intercomparison of multi-copy techniques, this study illustrates the usefulness of multi-copy chromosomal integration methods in caffeic acid production in S. cerevisiae, hoping to provide more reference and build a foundation for future development of heterologous biosynthesis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

HQ and YZL conceived and designed the experiments. HQ performed the experiments, wrote, and edited the manuscript. LY, MC, JZH, JYL, and LYH assisted in experiments. HJX and MQQ contributed materials and revised the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.